JP2018516572A - Methods and compositions for the treatment of RNA-induced HIV infection - Google Patents

Abstract

  Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) related endonucleases and specific target sequences in retroviruses that contain a nucleic acid encoding a guide RNA sequence complementary to one or more target nucleic acid sequences in the retroviral genome A composition for cutting.

Description

(Statement about federally sponsored research)
This invention was made with federal support with grants from the National Institutes of Health to Kamel Khalili (P30MH092177) and Wenhui Hu (R01NS087971).

  The present invention relates to compositions and methods for specifically cleaving a target sequence in a retrovirus (eg, human immunodeficiency virus (HIV)). Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -related endonuclease and a guide RNA sequence complementary to the target sequence in human immunodeficiency virus, the composition may be subject to HIV infection or at risk of HIV infection Can be administered.

  For over 30 years since the discovery of HIV-1, AIDS has been a major public health problem affecting over 35.3 million people worldwide. AIDS remains refractory due to the permanent integration of HIV-1 into the host genome. Current therapies to control HIV-1 infection and slow the progression of AIDS (highly active antiretroviral therapy (ie, HAART)) are responsible for viral replication in cells that maintain HIV-1 infection. Greatly suppresses viremia in plasma to a minimum level. However, HAART is unable to suppress low levels of viral genome expression and replication in tissues, and cells undergoing latent infection that serve as reservoirs for HIV-1 (eg, non-dividing memory T cells, brain Macrophages, microglia and astrocytes) cannot be targeted. Persistent HIV-1 infection has also been associated with co-morbidities including heart disease, kidney disease, osteopenia and neurological disease. There is an ongoing need for curative treatment strategies that target a permanent viral reservoir.

  Current therapies to control HIV-1 infection and prevent AIDS progression have dramatically reduced viral replication in cells susceptible to HIV-1 infection, but the HIV-1 proviral DNA Low levels of viral replication cannot be excluded in cells undergoing latent infection that contain an integrated copy. There is an urgent need for the development of curative therapeutic strategies that target permanent viral reservoirs, including strategies for the elimination of proviral DNA from the host genome.

  Recently, several novel systems for eliminating exogenous genes (homing endonuclease (HE), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR-related system 9 (Cas) protein Are being developed).

  In the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) method, a gene editing complex (comprising a guide RNA (gRNA) complementary to a Cas9 nuclease and a target viral DNA sequence) is constructed. The gRNA induces a Cas9 nuclease to bind to and cleave the viral DNA strand containing the target sequence. The Cas9 / gRNA gene editing complex introduces one or more mutations into the viral DNA.

  The possibility of genetically disrupting the integrated HIV-1 provirus using HE to target conserved viral protein sequences has been reported. ZFN targeting the co-receptor CCR5 of the HIV-1 host is in phase 2 clinical trials for the treatment of HIV / AIDS. TALEN has been shown to efficiently cleave the expected site CCR5. The Cas9 / gRNA editing complex has also been used to disrupt HIV-1 entry co-receptors (CCR5, CXCR4) and proviral structural proteins (Manjunath et al., Viruses, 14; 5 (11) : 2748-2766 (2013); Stone et al., Curr. Opin.HIV AIDS. 8 (3): 217-223 (2013); Wang et al., PLoS One.26; 9 (12): e115987 (2014 )). However, CCR5 is not the only receptor for HIV-1 infection and has many other cellular functions as well.

  The present invention provides compositions and methods relating to the treatment and prevention of retroviral infections, particularly human immunodeficiency virus HIV. The compositions and methods attack proviral HIV that is integrated into the genome of a human host.

  In particular, the invention relates to a nucleic acid sequence encoding a CRISPR-related endonuclease and one or more gRNAs that encode a plurality of gRNAs, each gRNA being complementary to a target sequence in the retroviral genome. Compositions comprising the isolated nucleic acid sequences are provided. In a preferred embodiment, two gRNAs are included in the composition, each gRNA induces a Cas endonuclease at a different target site that may be in the integrated retroviral DNA HIV DNA. DNA extending between the cleavage sites is deleted, resulting in excision of part or all of the HIV genome. Most effective combinations of multiple gRNAs are pairs where one gRNA targets a site in the LTR region and the other gRNA targets a site in a structural gene (eg, Gag or Pol) and both gRNAs in the LTR Contains pairs that target the site.

  The invention also provides a method of inactivating a retrovirus in a mammalian cell by exposing the cell to a composition comprising one or more isolated nucleic acids encoding a gene editing complex. . The gene editing complex includes a CRISPR-related endonuclease and one or more gRNAs, each gRNA being complementary to a target sequence in a retrovirus.

  The invention further provides a pharmaceutical composition for inactivation of integrated retroviral proviral DNA in a mammalian subject. The composition includes an isolated nucleic acid sequence encoding a Cas endonuclease and at least one gRNA that is complementary to a target sequence in proviral retroviral DNA (eg, HIV DNA). Pairs of gRNAs that target different sites in the retroviral genome are preferred. The isolated nucleic acid sequence is contained in at least one expression vector.

  The present invention further provides a method of treating a mammalian subject infected with a retrovirus (eg, HIV). The method includes determining that the mammalian subject is infected with HIV, administering an effective amount of the pharmaceutical composition described above, and treating the mammalian subject for HIV infection.

  The present invention also provides a method of treatment to reduce the risk of retroviral (eg, HIV) infection in a mammalian subject at risk of infection. The method includes determining that the mammalian subject is at risk of HIV infection, administering an effective amount of the pharmaceutical composition described above, and reducing the risk of HIV infection in the mammalian subject.

  The present invention further provides kits for the treatment or prevention of HIV infection. The kit includes at least one isolated nucleic acid sequence encoding a CRISPR-related endonuclease, and one or more gRNAs that are complementary to a target site in HIV. Alternatively, the kit includes one or more vectors encoding the nucleic acid. The kit also includes packaging materials, package inserts with instructions for use, sterile liquid, syringes and / or sterile containers.

Streptococcus pyogenesno CAS9, a single guide RNA (sgRNA) and a protospacer flanking motif (based on PAM NNG or NAG and sgRNA sequences, indicating cleavage at the third nucleotide of both strands of DNA ( PAM) is shown. The sgRNA is composed of CRISPR RNA containing a 20 bp spacer (seed or target sequence) and a 12 bp stem loop (GAAA) and a transactivated cRNA (tracRNA). Gene maps for spCAS9 expressing lentiviral vectors (top) and sgRNA expressing lentiviral vectors (bottom) are shown. 3 × Flag for immunodetection, reporter red fluorescent protein (RFP) for easy tittering and FACS analysis, and T2A peptide for self-cleavage to prevent the potential impact of RFP on CAS9 function The expressing lentiviral reporter vector (top) was purchased from Biosettia. BsI cloning site and antibiotic selection marker A sgRNA-expressing lentiviral vector was modified from the Addgene vector (# 50946), showing purromycin and reporter blue fluorescent protein (BFP) for easy tittering and FACS analysis. Figure 2 shows a diagram of the HIV-1 genome showing selected gRNAs targeting the HIV-1 LTR, Gag (AD) and Pol (A, B) regions. A detailed view of sgRNA targeting 400 bp within the U3 region of the HIV-1 LTR is shown. Seed sequences with green PAM (underlined bold) in the sense strand and red PAM (underlined bold) in the antisense strand are represented as shown. Most of them can be paired for Cas9 nickase and RNA-induced FokI nuclease (RFN), reducing potential off-target effects up to 1500-fold. Selection of the 400 bp region was based on the absence of all commonly used lentiviral vectors used for delivery of the gene and sgRNA. Such selection prevents self-cleavage of the lentiviral vector by Cas9 / gRNA. Includes enhanced firefly luciferase (ELuc), it shows a diagram of EcoHIV reporter vectors derived from human _{HIV NL4-3} vector. The eLuc gene was inserted between Env and Nef using a self-cleaving 2A peptide in front of Nef, replacing gp120 of the HIV-1 with gp80 from a species-directed murine leukemia virus. Figure 4 shows a single sgRNA screen by EcoHIV-luciferase reporter assay. HEK293T cells were cotransfected with the EcoHIV-cLuc reporter, pLV-EF1a-spCas9-T2A and the indicated sgRNA expression lentiviral vector. Two days later, the luciferase activity in the cell lysate was measured using the ONE-GLO® Luciferase Assay System. Data represent the mean ± 4 of 4 independent transfections. It is shown that Gag-D sgRNA paired with any of the LTR-sgRNAs reduced luciferase activity by 64-96%. HEK293T cells were cotransfected with the EcoHIV-cLuc reporter, pLV-EF1a-spCas9-T2A and the indicated sgRNA expression lentiviral vector. Two days later, the luciferase activity in the cell lysate was measured using the ONE-GLO® Luciferase Assay System. Data represent the mean ± 4 of 4 independent transfections. It shows that either Gag-sgRNA or LTR-3mpsgRNA paired with Pol-sgRNA reduced luciferase activity by 73-93%. HEK293T cells were cotransfected with the EcoHIV-cLuc reporter, pLV-EF1a-spCas9-T2A and the indicated sgRNA expression lentiviral vector. Two days later, the luciferase activity in the cell lysate was measured using the ONE-GLO® Luciferase Assay System. Data represent the mean ± 4 of 4 independent transfections. Figure 4 shows a single sgRNA screen by EcoHIV-luciferase reporter assay. HEK293T cells were cotransfected with the EcoHIV-cLuc reporter, pLV-EF1a-spCas9-T2A and the indicated sgRNA expression lentiviral vector. Two days later, the luciferase activity in the cell lysate was measured using the ONE-GLO® Luciferase Assay System. Data represent the mean ± 4 of 4 independent transfections. It shows that PCR genotyping has demonstrated the elimination of HIV-1 DNA between the 5'-TLR target site and the Gag-D cleavage site. Upper panel: position of PCR primers. Lower panel: GagD sgRNA paired with various sgRNAs targeting the LTR. HEK293T cells were cotransfected with the EcoHIV-cLuc reporter vector, the pLV-EF1a-spCas9-T2A vector and the indicated sgRNA expression lentiviral vector. Two days later, cells were lysed with 50 mM NaOH for 10 min at 95 ° C. and neutralized with 1 M Tris-HCl. The crude extract was used directly for PCR using Terra PCR Direct Polymerase Mix (Clontech) and PCR primers T361 / T458 directed to 5'-LTR and 5'-partial Gag (1364 bp). The PCR primer was used in a control sample transfected with an empty sgRNA expression vector. A 35 kb fragment is generated. It shows that PCR genotyping has demonstrated the elimination of HIV-1 DNA between the 3'-LTR target site and the Gag-D cleavage site. Upper panel: position of PCR primers. Lower panel: GagD sgRNA paired with various sgRNAs targeting the LTR. HEK293T cells were cotransfected with the EcoHIV-cLuc reporter vector, the pLV-EF1a-spCas9-T2A vector and the indicated sgRNA expression lentiviral vector. Two days later, cells were lysed with 50 mM NaOH for 10 min at 95 ° C. and neutralized with 1 M Tris-HCl. The crude extract was subjected to Terra PCR Direct Polymerase Mix (Clontech) and PCR primer T758 (796-817 nucleotides) / T645 targeting the entire 3′-LTR and HIV-1 genome (excluding some 5 ′ Gag sequences). Used directly for PCR with (targeting the vector sequence behind the 3 ′ LTR). The PCR primer produces a predicted 9.5 kb fragment that is undetectable by routine PCR conditions in a control sample transfected with an empty sgRNA expression vector. It shows that PCR genotyping has demonstrated the elimination of HIV-1 DNA between the LTR-3 target site and the Gag or Pol target site. Upper panel: position of PCR primers. Lower panel: GagD paired with LTR-1, 2, 3 or TLR-3 sgRNA paired with various sgRNAs targeting Gag or Pol. HEK293T cells were cotransfected with the EcoHIV-eLuc reporter vector, the pLV-EF1a-spCas9-T2A vector and the indicated sgRNA expression lentiviral vector. Two days later, cells were lysed with 50 mM NaOH for 10 min at 95 ° C. and neutralized with 1 M Tris-HCl. The crude extract is targeted to Terra PCR Direct Polymerase Mix (Clontech) and 5′-LTR and 5′-genomic sequences (left panel), or 3′-genomic and 3′-LTR (right panel) sequences. Used directly for PCR with PCR primers. Shown is a diagram of the location of PCR primers and Gag / Pol target sites used for valid sgRNA verification by PCR genotyping. Blot showing the results of GagD paired with various LTR-gRNAs. Blot showing the results of GagD paired with various LTR-gRNAs. Blot showing results of LTR-R paired with various gRNAs targeting Gag and Pol. Deletion of 5'LTR-Gag or Gag-3'LTR was detected. Wild-type (WT) band depth, deletion and insertion were quantified using NIH Image J. The numbers below the gel are the WT band change (%) relative to the empty rRNA control after normalization with the Cas9 PCR product using Cas9-specific primers T477 / T478, as well as the insertion band compared to the WT band. And the fold change of the deletion band. The dramatic changes induced by the corresponding gRNA are highlighted as multiple boxes. Blot showing results of LTR-R paired with various gRNAs targeting Gag and Pol. Deletion of 5'LTR-Gag or Gag-3'LTR was detected. Wild-type (WT) band depth, deletion and insertion were quantified using NIH Image J. The numbers below the gel are the WT band change (%) relative to the empty rRNA control after normalization with the Cas9 PCR product using Cas9-specific primers T477 / T478, as well as the insertion band compared to the WT band. And the fold change of the deletion band. The dramatic changes induced by the corresponding gRNA are highlighted as multiple boxes. FIG. 6 shows further validation of effective gRNA by direct PCR genotyping with additional PCR primers directed to the structural region and the 3′-LTR. FIG. 3I shows a pair of forward primer (T758) for the 5 'upstream of gRNA GagD with reverse primer (T645) for the vector downstream of the 3' terminal LTR. The arrow represents a non-specific band (ns). FIG. 6 shows further validation of effective gRNA by direct PCR genotyping with additional PCR primers directed to the structural region and the 3′-LTR. FIG. 3J shows a pair of forward primers for the 5 ′ upstream of gRNA GagD with the reverse primer (T422) for the R region of the 3 ′ terminal LTR. The arrow represents a non-specific band (ns). It shows that representative TA cloning and Sanger sequencing confirmed a 296 bp deletion between LTR-1 and LTR-3 and an additional 180 bp insertion between the two cleavage sites. Sample preparation and Direct PCR were performed as described in FIG. 3A. The PCR fragment after cleavage was extracted for TA cloning and Sanger sequence. The red arrow points to the expected cleavage site at the third nucleotide from PAM. Underlined red indicates PAM sequence. FIG. 5A includes successful targeting strategies and predicted results of multiple viral LTR sequences (flanking, integrated provirus) using the HIV-1 genome (Cas9 / gRNA complex) )) Is a schematic explanation. The detailed structure of LTR is shown. The sequence of target sites and their positions in the LTR are shown. Figure 2 shows a diagram of the Jurkat 2D10 reporter cell line (including a depiction of the integrated HIV-1 reporter sequence). FIG. 5 shows the microscopic chain truth of PMA / TSA-induced reactivation of the proviral sequence during the incubation period. FIG. 5 shows a flow cytometry histogram representing PMA / TSA-induced reactivation of the latent proviral sequence. The results of screening a single cell clone are shown. It shows the confirmation of the best clone according to the flow cytometry of the clone obtained in the screening of a single cell clone. Shown are exemplary Western blots for FLAG-Cas9 (upper panel) and RT-PCR agarose gel electrophoresis (lower panel) for gRNA expression. The location of the primers used for PCR analysis of HIV-1 provirus exclusion from the host genome is indicated. Each primer was specific for the proviral Env gene sequence motif (RRE), the genomic sequence flanking the integrated reporter provirus (chromosome 16, MSRB1 gene), the LTR and the control b-actin gene. A photograph of an agarose gel of a PCR reaction is shown. The arrow points to the part of the band that disappears due to exclusion of the HIV-1 sequence. Extensive PCR data is shown in conditions that are optimized for shorter products (allowing detection of proviral lariat sequences at the integration site). The sequencing results for provirus lariat are shown. FIG. 5 shows a fluorescence analysis of a Jurkat 2D10 single cell clone infected with HIV-1 NL-4-3-EGFP-P2A-Nef, the course of infection being monitored over 18 days in the method of fluorescence analysis. Figure 2 shows a Western blot showing Cas9-FLAG expression in the tested clones. Figure 5 shows agarose gel pictures of reverse transcription PCR for gRNA expression in selected clones. FIG. 2 shows a fluorescence micrograph of Jurkat 2D10 cells transduced with lentivirus expressing RFP-Cas9 and / or LTR A / B ′ gRNA. FIG. 5 shows flow cytometric analysis of RFP-Cas9 and / or LTR A / B ′ gRNA expression after induction with PMA / TSA. The sequences of LTR A and LTR B 'are shown. FIG. 6 shows Surveyor assay analysis of off-target indels after expression of Cas9 and LTR A / B ′. The results of Sanger sequencing analysis of off-target indels are shown. The position of the HIV-1 reporter integration site in the second exon of the MSRB1 gene on chromosome 16 and the adjacent gene is shown. The results of qRT-PCR comparison of the expression of adjacent genes in control and Cas9 / LTR AB 'expressing cells are shown. Diagram of representative sample TA cloning and Sanger sequencing confirming the expected deletion of fragments between the corresponding gRNA target sites (AC) and various additional insertions (FIGS. 12B, C). Is shown. The PAM sequence is highlighted and scissors represent the third nucleotide from PAM. The arrow points to the ligation site after cleavage and ligation. It shows the establishment of a stable EcoHIV-firefly luciferase HEK293T cell line. EcoHIV-eLuc virus was harvested 48 hours after cotransfection of HEK293T cells in 1 well of a 6-well plate using 3 μg of pEcoHIV-eLuc plasmid and 1 μg of VSV-G plasmid. An equal volume of virus supernatant (250 μl) was added to HEK293T cells (2 × 10 ⁴ / well) in 12 wells. After 48 hours, ELuc luciferase activity was measured using the ONE-Glo Luciferase Assay (Promega), from the luciferase activity in the control HEK293T cells without viral treatment, it showed a 10 ⁵ fold higher luciferase activity. A single cell was then limiting diluted and cultured in four 96-well plates. After 2-3 weeks, surviving cell colonies were isolated and eLuc reporter activity (FIGS. 13A, B) and primers T361 (5′-gatctgtggatctaccacacacaaca-3 ′) and T458 (5′-cccactgtgtttagcatggtatt) by ONE-Glo luciferase assay. -3 ') was used to test for confirmation of the EcoHIV-eLuc transgene by Direct-PCR. Half of the 13 clones from one cell in the first round of experiments (A, number 1016) and half of the 20 clones in the second round (B, 1021) have varying degrees of constant eLuc activity. showed that. Two of them (1016-11 and 1021-19) were significantly responsive to stimulation with TNFα (10 ng / ml) and PMA (10 ng / ml). All eLuc expressing clones (FIGS. 13C-E) contained the transgene except for clone 1021-6 (green square). Two clones (1016-6 and 1016-9) contained the transgene but did not show eLuc activity even after treatment with latency reversal agents such as TNFα / PMA. Clone 1021-6 appears to have lost the Gag sequence during integration, but still transcribes eLuc via alternative splicing. Therefore, these clones having eLuc activity were selected and maintained for further testing. Some of these clones could not be passaged due to the continued production of virulent viral proteins, and some clones resisted transfection even using the Lipofectamine kit. The results of the effectiveness screening of gRNA using the stable EcoHIV-firefly luciferase HEK293T cell line are shown. 14A, B: EcoHIV-eLuc stable expression clones were infected with 10 MOI of pCW-Cas9-puromycin lentivirus and selected with puromycin (1 μg / ml) for 2 weeks. Two days later, the ONE-Glo luciferase assay was performed. FIGS. 14, 14D: EcoHIV-eLuc stably expressing cells were co-transfected with the indicated gRNA expression vector and the LV-EF1α-spCas9-T2A-RFP vector. Luciferase activity was measured after 48 hours using the ONE-Glo luciferase assay. The data represents the mean ± SEM of four independent transfections for the percent change in eLuc activity when compared to the empty gRNA ZERO group.

  In part, the present invention relates to a Clustered Regularly Interspace Short Palindromic Repeat (CRISPR) -Cas9 nuclease system (Cas9 / C), in which an integrated human immunodeficiency virus (HIV) genome is guided by RNA in one or more configurations. gRNA) is based on the discovery that it can be removed from HIV-infected cells.

(Definition)
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any method and material similar or equivalent to those described herein can be used in the practice for testing the present invention. However, it is preferred to use the materials and methods described herein. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is used only for the purpose of describing particular embodiments and is not intended to be limiting.

  All genes, gene names, and gene products described herein are intended to correspond to homologues from any species to which the compositions and methods disclosed herein are applicable. Where a gene or gene product derived from a particular species is disclosed, this disclosure is intended only to be illustrative and should not be construed as limiting unless it can be taken from the context. I want you to understand. Thus, for example, reference to a gene or gene product disclosed herein is intended to encompass homologous and / or orthologous genes and gene products derived from other species.

  As used herein, the articles “a” and “an” refer to one or more (ie, at least one) grammatical objects of the article. By way of example, “an element” means one element or more than one element. Therefore, the description “acell” includes, for example, a plurality of cells of the same type. Also, the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are more detailed. To the extent used in the description and / or claims, such terms are intended to be inclusive, as well as the term “comprising”.

  As used herein, the terms “comprising”, “comprise” or “comprised” and variations thereof are related items, compositions, apparatus, methods, processes, Meaning that a defined or described element, such as system, is inclusive or open-ended. These may include additional elements, and the defined or described items, compositions, apparatus, methods, processes, systems, etc., include those specified elements (or their equivalents as appropriate). , Indicates that other elements may be included. It also indicates that the inclusion of other elements still falls within the scope / definition of defined items, compositions, devices, methods, processes, systems, etc.

  In this document, “about” as used with reference to measurable values (such as amount or length of time) is ± 20%, ± 10%, ± 5%, ± 1% from a particular value, or It is meant to include a variation of ± 0.1%. This means that the variation value is suitable for carrying out the method of the present disclosure. Alternatively, particularly with respect to biological systems or processes, the term can also mean values on the order of 5 times and on the order of 2 times. Where specific values are recited in the present application and claims, the term “about” shall mean within an acceptable error range for the particular value, unless otherwise specified.

  “Coding” refers to the intrinsic nature of a particular sequence of nucleotides in a polynucleotide (such as a gene, cDNA, or mRNA). The above properties include (i) the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (ie, rRNA, tRNA, and mRNA) or a defined sequence of amino acids. And (ii) the biological properties resulting from this. Thus, if transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, it can be said that the gene encodes the protein. Both the coding strand (its nucleotide sequence is identical to the mRNA sequence and is usually listed in the sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) Can be said to encode a protein or other product.

  An “effective amount” or “therapeutically effective amount” of a compound is that amount of the compound sufficient to produce a beneficial effect in the subject to which it is administered. An “effective amount” of a delivery vehicle is an amount sufficient to effectively bind or deliver the compound.

  As used herein, the term “exclusion” of a virus (eg, HIV) means in vivo by preventing the virus from replicating, preventing the virus from migrating or infecting any other cell or subject. Means the disappearance, fragmentation, degradation, genetic inactivation, or any other physical, biological, chemical or structural manifestation of the genome from which the virus is removed. In some cases, fragments of the viral genome may be detectable, but the virus cannot replicate or infect.

  “Expression vector” refers to a vector comprising a recombinant polynucleotide having expression control sequences operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression. Other elements for expression may be supplied by the host cell or may be present in an in vitro expression system. Expression vectors include anything known in the art. Examples include cosmids, plasmids (eg, neat or stored in liposomes), and viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides, etc. Is mentioned.

  “Homologous” refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit (eg when each of the two DNA molecules is occupied by adenine), the molecule is in that position Homologous. The percent homology between two sequences is a function of x100, divided by the number of positions compared, the number of matching or homologous positions shared by the two sequences. For example, if 6 out of 10 positions in two sequences are identical or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. In general, the comparison is performed with the two sequences aligned to give the greatest homology.

  “Isolated” means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally occurring in an animal's body is not “isolated”, but the same nucleic acid or peptide that is partially or completely separated from its naturally occurring coexisting material is “isolated”. It has been done. An isolated nucleic acid or protein may exist in substantially purified form, or may exist in a non-native environment (eg, a host cell).

  In the context of the present invention, for ordinary nucleobases, the following abbreviations are used: “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, and “T” refers to thymidine. "U" refers to uridine.

  Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are in degenerate versions of each other and that encode the same amino acid sequence. Also, the term “nucleotide sequence encoding a protein or RNA” may include introns to the extent that several versions of the nucleotide sequence encoding the protein may include introns.

  The terms “patient”, “subject”, “individual” and the like are used interchangeably herein and refer to any animal or cell (in vitro or in situ) to which the methods described herein can be applied. In certain non-limiting embodiments, the patient, subject or individual is a human.

  “Parenteral” administration of the composition involves, for example, injection or infusion techniques, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal. Contains.

  As used herein, the terms “polynucleotide”, “nucleic acid sequence” and “gene” are used interchangeably throughout this specification to refer to complementary DNA (cDNA), natural and / or modified monomers or linkages. Linear or cyclic oligomers or polymers (including deoxyribonucleosides, ribonucleosides, substituted and alpha anomeric forms thereof, peptide nucleic acids (PNA), lock nucleic acids (LNA), phosphorothioates, methylphosphonates, etc.) Is included. As used herein, the term “polynucleotide” is defined as a chain of nucleotides. In addition, nucleic acids are nucleotide polymers. Thus, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have the common knowledge that nucleic acids are polynucleotides and that the polynucleotides can be hydrolyzed into monomeric “nucleotides”. Monomeric nucleotides can be hydrolyzed to nucleosides. A polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art. Such means include recombinant means (ie, means for cloning nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR ™, etc.), and synthetic means, It is not limited to. The nucleic acid sequence can be “chimeric”. That is, it can be composed of different regions. In the context of the present invention, a “chimeric” compound is an oligonucleotide that contains two or more chemical regions (eg, DNA region, RNA region, PNA region, etc.). Each chemical region is composed of at least one monomer unit (ie, nucleotide). These sequences typically include at least one region, which has been modified to exhibit one or more desired properties.

  An “analog” with respect to a nucleoside is a synthetic nucleoside having a modified base moiety and / or a modified sugar moiety (eg, Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid. Res. , 1997, 25 (22), 4429-4443, Toulme, JJ, Nature Biotechnology 19: 17-18 (2001); ManoharanM., Biochemica et Biophysica Acta 1489: 117-139 (1999); FreierS. M., Nucleic Acid Research, 25: 4429-4443 (1997), Uhlman, E., Drug Discovery & Development, 3: 203-213 (2000), Herdewin P., Antisense & Nucleic Acid Drug Dev., 10: 297-310 (2000) 2′-O, 3′-C-linked [3.2.0] bicycloarabinonucleosides (eg NK Christiensen., Etal., J. Am. Chem. Soc., 120: 5458-5463 (See 1998))). Such analogs include synthetic nucleosides that are designed to improve binding properties (eg, 2-fold or 3-fold stability or specificity).

  The term “variant” when used in the context of a polynucleotide sequence may encompass a polynucleotide sequence associated with a wild-type gene. This definition may also include, for example, “allelic” variants, “splice” variants, “species” variants, or “polymorphic” variants. A splice variant may have significant identity to the referencing molecule, but generally has a greater or lesser number of polynucleotides. This is because different exons are spliced during mRNA processing. Corresponding polypeptides can have additional functional domains or have a domain loss. A species variant is a polynucleotide sequence that differs between one species and another. Variants of wild type gene products are particularly useful in the present invention. A variant may result from at least one mutation in the nucleic acid sequence, resulting in a different mRNA or polypeptide (these structures or functions may or may not be altered). Any natural or recombinant gene may have no allelic form, or one or more allelic forms. Common mutations that cause variants are generally due to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes can occur in a given sequence one or more times, alone or in combination with others.

  Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are in degenerate versions of each other and that encode the same amino acid sequence. The term “nucleotide sequence encoding a protein or RNA” may include introns to the extent that several versions of the nucleotide sequence encoding the protein may include introns.

  As used herein, the terms “peptide”, “polypeptide”, and “protein” are used interchangeably and mean a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide essentially contains at least two amino acids, and the maximum number of amino acids that can be included in a protein sequence or peptide sequence is not limited. Thus, for example, the terms oligopeptide, protein and enzyme may be produced using recombinant techniques, chemical or enzymatic synthesis, or may be naturally occurring polypeptides or peptides. Included in the definition of Polypeptide includes any peptide or protein having two or more amino acids joined to each other by peptide bonds. As used herein, the terms include short chains (usually referring to peptides, oligopeptides and oligomers, for example, in the field of the present invention) and long chains (in the field of the present invention many types Refers generally to a protein). "Polypeptide" refers to, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins Is included. The term also includes polypeptides that have been modified or derived (especially by glycosylation, acetylation, phosphorylation, etc.). Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

  As used herein, a “variant” of a polypeptide refers to an amino acid sequence that has been altered by one or more amino acid residues. A variant may have “conservative” changes (eg, replacing leucine with isoleucine) in which the substituted amino acid has similar structural or chemical properties. More rarely, a variant may have “nonconservative” changes (eg, replacing glycine with tryptophan). Analog minor variations can also include amino acid deletions or insertions, or both. Guidance on determining which amino acid residues can be substituted, inserted or deleted without destroying biological activity can be found using computer programs known in the art (eg, LASERGENE software (DNASTAR)). Good.

  A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of a medical condition for the purpose of reducing or eliminating those signs.

  As used herein, the phrase “therapeutically effective amount” refers to preventing or treating a disease or abnormality (delaying or preventing the onset of the disease or abnormality, preventing, inhibiting, reducing the progression of the disease or abnormality). Refers to an amount that is sufficient or effective to reduce (including relieve symptoms of such disease).

  “Treatment” is an intervention performed with the purpose of preventing the development or change of a disease state or symptom of a disease. “Treatment” thus refers to both therapeutic treatment and prophylactic or preventative measures. A “treatment” may be specified as palliative care. Those who need treatment include those who already have a disease and those who want to prevent the disease. Thus, “treating” or “treatment” of a condition, illness or abnormality is (1) the clinical or subclinical status of the condition, illness or abnormality that is suffering from or predisposed to the condition, illness or abnormality. Preventing or delaying the appearance of a clinical symptom of the condition, disease or abnormality that develops in a human or other mammal who has not experienced or manifested clinical symptoms; (2) inhibits the condition, disease or abnormality To suppress, reduce, or delay disease development or its recurrence (in the case of continuous treatment) or at least one clinical or subclinical symptom thereof; or (3) alleviate the disease I.e. causing at least one regression of the condition, disease or abnormality or clinical or subclinical symptoms thereof Succoth, it contains. The benefit to the individual being treated is statistically significant or at least perceivable by the patient or physician.

  A “vector” is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid compounds and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

  Scope: Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity. Moreover, such a description should not be construed as an unchangeable limitation on the scope of the invention. Accordingly, the description of a range should be considered to include not only individual numerical values within that range but also all possible subranges specifically disclosed. For example, the description of a range from “1 to 6” includes not only individual numerical values within the range (eg, 1, 2, 2.7, 3, 4, 5, 5.3, and 6), but also a specific Should also be considered to include subranges disclosed in (1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.). This applies regardless of the breadth of the range.

  Where any amino acid sequence is specifically referred to by a Swiss Prot or GENBANK accession number, that sequence is incorporated into this disclosure by reference. Information related to accession numbers (such as signal peptide, extracellular domain, transmembrane domain, promoter sequence, and translation start point identification) are all incorporated by reference into this disclosure.

(CRISPR-Nucleic acid composition)
Application of Cas9 technology (especially targeting the LTR) in eliminating HIV-1 harboring hosts has been shown to be a promising strategy for treatment or therapy of AIDS. Hu, et al., PNAS 2014, 111: 114616 shows that stable transfection of human cell cultures using a plasmid expressing Cas9 / gRNA targeted to a site in the HIV-1 LTR is responsible for host cell function. It has been disclosed that a portion and / or whole of the HIV-1 genome has been successfully eliminated without weakening. The target sites were called LTR-A, LTR-B, LTR-C and LTR-D. Targeting two different sites in the LTR was particularly effective in generating deletions that were sufficiently large to form all or substantially all removal of the proviral DNA sequence. The pre-existence of Cas9 / gRNA in the cells also prevented new HIV-1 infection.

  Because HIV and other retroviruses are highly susceptible to mutation, Cas9 / gRNA reagents and methods for targeting the integrated HIV genome require a broad spectrum. For certain uses, Cas9 / gRNA reagents effectively target HIV structural genes such as gag and pol.

  Accordingly, embodiments of the invention relate to compositions and methods for the treatment and elimination of highly susceptible and / or latent viruses from host cells in vitro or in vivo. The methods of the invention can be used to remove viruses or other foreign genetic material from a host organism without interfering with the integrity of the host's genetic material. Nucleases can be used for target viral nucleic acids. Therefore, it prevents viral replication or transcription or even deletes the viral genetic material from the host genome. Nucleases are specifically targeted to remove only viral nucleic acids when they are present as particles in the cell or when they are integrated into the host genome without affecting the host material. Can be done. Targeting the viral nucleic acid can be performed using a sequence specific portion (such as a guide RNA that targets viral genomic material for nuclease disruption but does not target the host cell genome). In some embodiments, CRISPR / Cas nuclease and guide RNA (gRNA) (both targeting and selectively editing or destroying viral genomic material) are used. CRISPR (clustered regularly interspaced shortpalindromic repeats) is a naturally occurring element of the bacterial immune system that protects bacteria from phage infection. Guide RNA localizes the CRISPR / Cas complex to the viral target sequence. Binding of the complex localizes the Cas endonuclease to the viral genome target sequence and causes disruption in the viral genome. Can be used to degrade or inhibit viral nucleic acids without interfering with the normal function of other nuclease systems (eg, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, or host genetic material Any other system can be used).

  The compositions embodied herein can be used to target viral nucleic acids in any form or at any stage in the viral life cycle. The targeted viral nucleic acid can be present in the host cell as an independent particle. In preferred embodiments, the viral infection is latent and the viral nucleic acid is integrated into the host genome. Any suitable viral nucleic acid can be a target for cleavage and digestion.

  CRISPR / Cas system: The CRISPR / Cas system is a gene editing complex containing a CRISPR-related nuclease (eg Cas9) and a target sequence located on the DNA strand (target in proviral DNA integrated in the mammalian genome) A guide RNA complementary to the sequence). An exemplary gene editing complex is shown in FIG. 1A. The gene editing complex can cleave DNA without a target sequence. And then this cleavage can cause the introduction of various mutations into the proviral DNA, leading to inactivation of the HIV provirus. The mechanism by which such mutations inactivate proviruses can vary. For example, mutations can affect proviral replication and viral gene expression. Mutations may be located in regulatory or structural gene sequences, resulting in incomplete production of HIV. Mutations can include deletions. The size of the deletion can vary from 1 nucleotide base pair to about 10,000 base pairs. In some embodiments, the deletion can include all or substantially all of the integrated retroviral nucleic acid sequence. Mutations can include insertions (ie, the addition of one or more nucleotide base pairs to the proviral sequence). The size of the inserted sequence can also vary (eg, from about 1 base pair to about 300 nucleotide base pairs). Mutations can include point mutations (ie, one nucleotide is replaced with another nucleotide). Useful point mutations are those that have a functional result (eg, a mutation that changes an amino acid codon to a stop codon or a mutation that results in the production of a non-functional protein).

  In general, CRISPR / Cas proteins contain at least one RNA recognition and / or RNA binding domain. The RNA recognition and / or RNA binding domain interacts with the guide RNA. A CRISPR / Cas protein can also include a nuclease domain (ie, DNase domain or RNase domain), DNA binding domain, helicase domain, RNase domain, protein-protein interaction domain, dimerization domain, and other domains.

  In embodiments, the CRISPR / Cas-like protein can be a wild-type CRISPR / Cas protein, a modified CRISPR / Cas protein, or a fragment of a wild-type or modified CRISPR / Cas protein. CRISPR / Cas-like proteins can be modified to increase nucleic acid binding affinity and / or specificity, change enzyme activity, and / or change other properties of the protein. For example, the nuclease (ie DNase, RNase) domain of a CRISPR / Cas-like protein can be modified, deleted or inactivated. Alternatively, CRISPR / Cas-like proteins can be truncated to remove domains that are not essential for the function of the fusion protein. CRISPR / Cas-like proteins can be truncated or modified to optimize the activity of the effector domain of the fusion protein.

  In embodiments, the CRISPR / Cas system can be a Type I, Type II, or Type III system. Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5 Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf, Csf3, Csf2, Csf3, Csf2, Csf

  In one embodiment, the RNA guide endonuclease is derived from a type II CRISPR / Cas system. In other embodiments, the RNA guide endonuclease is derived from a Cas9 protein. Cas9 protein, Streptococcus pyogenes, Streptococcusthermophilus, Streptococcus sp., Nocardiopsisdassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomycesviridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacilluspseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillusdelbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans , Polaromonassp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcussp., Acetohalobium arabaticum, Ammonifexdegensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridiumbotulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacilluscaldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseud oalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobiumevestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbyasp., Microcoleus chthonoplastes, P Can do.

  In some embodiments, the CRISPR / Cas-like protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the CRISPR / Cas-like protein can be derived from a modified Cas9 protein. For example, the amino acid sequence of a Cas9 protein can be modified to change one or more properties of the protein (eg, nuclease activity, affinity, stability, etc.). Alternatively, a domain of Cas9 protein that is not involved in RNA-guided cleavage can be removed from the protein such that the modified Cas9 protein is smaller than the wild-type Cas9 protein.

  An exemplary and preferred CRISPR-related endonuclease is Cas9 nuclease. The Cas9 nuclease can have the same nucleotide sequence as the wild type Streptococcus pyrogenes sequence. In some embodiments, the CRISPR-related endonuclease is other species (eg, other Streptococcus species (such as thermophilus), Pseudomonas aeruginosa, Escherichia coli, or other bacterial genomes and archeas that have been sequenced, or other It can be a sequence derived from a prokaryotic microorganism. Alternatively, the wild type Streptococcus pyrogenes Cas9 sequence can be modified. Nucleic acid sequences can be codons optimized for efficient expression in mammalian cells (ie, “humanized”). The humanized Cas9 nuclease sequence can be, for example, a Cas9 nuclease encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 GI: 669193757; KM099232.1GI: 669193761; or KM099233.1 GI: 669193765. It can be an array. Alternatively, the Cas9 nuclease sequence can be, for example, a sequence contained within a commercially available vector (PX330 or PX260 from Addgene (Cambridge, Mass.)). In some embodiments, the Cas9 endonuclease is a Genbank accession number KM099231.1 GI: 669193757; KM099232.1GI: 669193761; or KM099233.1 GI: 669193765 Cas9 endonuclease sequence or PX330 or PX260 (Addgene, Mass.). It may have an amino acid sequence that is a variant or fragment of any of the Cas9 amino acid sequences of Cambridgeshire. The Cas9 nucleotide sequence can be modified to encode a biologically active variant of Cas9, such as one or more mutations (eg, addition, deletion, or substitution mutations, or the like An amino acid sequence different from wild-type Cas9. The one or more substitution mutations can be substitutions (eg, conservative amino acid substitutions). For example, a biologically active variant of a Cas9 polypeptide has at least or about 50% sequence identity to a wild-type Cas9 polypeptide (eg, at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity). Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; Histidine and arginine; and phenylalanine and tyrosine. The amino acid residues in the Cas9 amino acid sequence can be non-naturally occurring amino acid residues. Naturally occurring amino acid residues include those naturally encoded by the genetic code, and nonstandard amino acids (eg, amino acids having a D-configuration instead of an L-configuration). The peptides can also include amino acid residues that are modified versions of standard residues (eg, pyrrolidine can be used in place of lysine and selenocysteine can be used in place of cysteine). Non-naturally occurring amino acid residues, which have never been found in nature, are composed of the basic formula of amino acids and can be incorporated into peptides. These include D-alloisoleucine (2R, 3S) -2-amino-3-methylpentanoic acid and L-cyclopentylglycine (S) -2-amino-2-cyclopentylacetic acid. For other examples, you can refer to textbooks or the World Wide Web (sites currently maintained by the California Institute of Technology list structures of unnatural amino acids that have been successfully incorporated into functional proteins) .

  The Cas9 nuclease sequence can be a mutated sequence. For example, Cas9 nuclease can be mutated in conserved HNH and RuvC domains (which are involved in chain-specific cleavage). For example, an aspartic acid to alanine mutation (D10A) in the RuvC catalytic domain causes the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA, resulting in single-strand breaks and subsequent HDR Further selective repair can potentially reduce the frequency of unwanted indel mutations from off-target double-strand breaks.

  The present invention incorporates several advantages over the Cas9 / gRNA system disclosed in Hu, et al, PNAS 2014, 111: 114616. In the experiments disclosed in the Examples, additional highly specific target sequences were identified both inside the HIV-1 LTR and inside the HIV-1 structural gene. These target sequences (also called target “sites”) have been efficiently edited by Cas9 / gRNA, resulting in viral gene expression and inactivation of replication in latently infected mammalian cells. Some of these additional Cas9 / gRNA constructs and combinations thereof have been found to result in the removal of all or part of the integrated HIV proviral DNA from the host cell genome. A pair of constructs with one component directed to the LTR target site and another component directed to the structural gene target site was particularly effective in producing removal or elimination of the HIV genome. This is the first demonstration that an integrated attack on the LTR site and the structural gene can result in the removal of an intervening stretch of integrated HIV DNA. The present invention therefore greatly expands the spectrum of Cas9 / gRNA compositions that can be used to target HIV DNA integrated in host cells.

  Accordingly, the present invention relates to an isolated nucleic acid sequence that encodes a CRISPR-related endonuclease and one or more gRNAs that are complementary to a target sequence in HIV or other retrovirus. Characterizing a composition for use in inactivating proviral DNA incorporated into a host cell, comprising a sequence.

  gRNA includes mature crRNA containing a unique target sequence (called a spacer) of about 20 base pairs (bp), and trans-activated small RNA that serves as a guide for ribonuclease III-mediated processing of pre-crRNA ( tracrRNA). The crRNA: tracrRNA duplex structure directs Cas9 to the target DNA through pairing of complementary bases between the spacer on the crRNA and a complementary sequence on the target DNA (called a protospacer). Cas9 recognizes the trinucleotide (NGG) protospacer adjacent motif (PAM) and identifies the cleavage site (the third nucleotide from PAM). In the present invention, crRNA and tracrRNA may be expressed separately or recombined as an artificial fusion gRNA via a synthetic stem loop (AGAAAU) to mimic the natural crRNA / tracrRNA duplex. It may be. Such gRNAs can be synthesized for direct RNA transfection or transcribed in vitro, or expressed from an RNA expression vector promoted to U6 or H1.

  In the compositions of the invention, each gRNA contains a sequence that is complementary to the target sequence in the retrovirus. An exemplary target retrovirus is HIV, but the compositions of the invention are also useful for targeting other retroviruses such as HIV-2 and simian immunodeficiency virus (SIV) -1.

  Some exemplary gRNAs of the invention are complementary to a target sequence in the long terminal repeat (LTR) region of HIV. The LTR is subdivided into a U3 region, an R region and a U5 region. The structure of the U3 region, R region and U5 region of HIV-1 is shown in FIG. 3A. The LTR contains all the signals necessary for gene expression and is involved in the integration of the provirus into the genome of the host cell. For example, in U3, a base or core promoter, core enhancer and regulatory region are found, while in R a transcription-promoting response element is found. In HIV-1, the U5 region is a plurality of subregions (eg, TAR or transcriptional response elements (involved in transcriptional activation); poly A (involved in dimerization and genomic packaging); PBS Or a primer binding site; Psi or packaging signal; DIS or dimer start site). Thus, in some embodiments, the gRNA target sequence comprises one or more target sequences within the LTR region of HIV proviral DNA and one or more within structural and / or nonstructural genes of HIV proviral DNA. Of target sequences. In other embodiments, the gRNA target sequence comprises one or more target sequences within the LTR region of HIV proviral DNA and one or more target sequences within the structural gene. In another embodiment, the gRNA target sequence comprises one or more target sequences in the LTR region of HIV proviral DNA and one or more target sequences in nonstructural genes of HIV proviral DNA. In yet another embodiment, the gRNA target sequence comprises one or more target sequences within the HIV proviral structural gene and one or more target sequences within the nonstructural gene of HIV proviral DNA. In yet another embodiment, the gRNA target sequence comprises one or more target sequences within the HIV proviral non-coding gene and one or more target sequences within the coding gene of HIV proviral DNA. In yet another embodiment, the gRNA target sequence comprises one or more target nucleic acid sequences in the first gene and one or more target nucleic acid sequences in the second gene; or One or more target nucleic acid sequences in the first gene and one or more target nucleic acid sequences in the third gene; or one or more target nucleic acid sequences in the first gene, One or more target nucleic acid sequences in the second gene and one or more target nucleic acid sequences in the third gene; or one or more target nucleic acid sequences in the second gene Including, one or more target nucleic acid sequences within the third gene or fourth gene; or any combination thereof. As can be seen, any combination of target nucleic acid sequences can be used and is only limited by the imagination of those skilled in the art.

  In the experimental results disclosed in the Examples, it was found that certain sequences within the U3, R and U5 regions of the LTR are useful target sequences. The gRNAs complementary to these target sequences are shown in FIGS. 1D and 3A of Example 2, FIG. 11A of Example 3, and Table 1 of Example 4. They are LTR 1, LTR 2, LTR 3, LTR A, LTR B, LTR B ', LTR C, LTR D, LTR E, LTR F, LTR G, LTR H, LTR I, LTR J, LTR K, LTR Includes L, LTR M, LTR N, LTR O, LTR P, LTR Q, LTR R, LTR S, and LTR T. The sequences of these gRNAs are shown in FIGS. 11A, 12A, 12B and 12C. The compositions of the present invention include, but are not limited to, these exemplary gRNAs, and can include gRNAs that are complementary to any suitable target site in the HIV LTR.

  Some of the exemplary gRNAs of the present invention target sequences in the HIV protein coding genome. It has been found that sequences within the gene encoding the structural protein gag are useful target sequences. Examples of gRNA complementary to these target sequences include GagA, Gag B, Gag C, and Gag D. Their target sites in the HIV-1 genome are shown in FIG. 3A and their nucleic acid sequences are shown in Table 1. Useful target sequences have also been found in the gene encoding the structural protein pol. Examples of gRNA complementary to these target sequences include PolA and PolB. Their target sites in the HIV-1 genome are shown in FIG. 3A and their nucleic acid sequences are shown in Table 1. The sequence for gRNA complementary to the target site in the HIV-1 envelope protein env is also shown in Table 1.

  Accordingly, the composition of the present invention includes these exemplary gRNAs, but is not limited thereto, HIV coding protein genes (structural protein tat, as well as accessory proteins vif, nef (negative factor) vpu (Virus protein U). ), Vpr, and tev may be included, but may include gRNA complementary to any suitable target site.

  The guide RNA sequence according to the present invention may be a sense sequence or an antisense sequence. Guide RNA sequences generally include a protospacer flanking motif (PAM). The sequence of the PAM can vary depending on the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system derived from S. pyogenes, the target DNA is typically immediately before the 5'-NGG protospacer adjacent motif (PAM). Therefore, for S. pyogenes Cas9, the PAM sequence can be AGG, TGG, CGG or GGG. Other Cas9 orthologs may have different PAM specificities. For example, Cas9 derived from S. thermophilus requires 5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3. Cas9 derived from Neiseria menigiditis requires 5'-NNNNNGATT. Although the specific sequence of the guide RNA can vary, regardless of the sequence, a useful guide RNA sequence is a retrovirus (genomically integrated) that achieves high efficiency while minimizing off-target effects ( For example, a sequence that completes the removal of HIV). The length of the guide RNA sequence is about 20 to about 60 nucleotides or more, for example, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, About 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 nucleotides, or It can be more than that. A useful screening method for identifying regions of very low homology between foreign viral genomes and host cell genomes (including endogenous retroviral DNA) is by bioinformatic screening using 12 bp + NGG target selection criteria. , Methods of excluding off-target human transcriptome or (although rarely) untranslated genomic sites; methods of avoiding transcription factor binding sites within the HIV LTR promoter (which is potentially conserved in the host genome) And methods for identifying and eliminating potential off-target effects by WGS, Sanger sequencing, and SURVEYOR assays.

  The guide RNA sequence may be configured as a single sequence, or may be configured as a combination of one or more different sequences (eg, multiple structures). The multiplex structure may comprise a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different guide RNAs.

  In the experiments disclosed in Examples 2 and 3, it was found that the gRNA combination is particularly effective when expressed in multiple ways (ie, simultaneously in the same cell). In many cases, the combination resulted in the removal of HIV provirus that spread between target sites. The removal can contribute to the elimination of the sequence between the breaks induced by Cas9 at each of the plurality of target sites. These combinations are pairs of gRNAs that comprise a component that is complementary to a target site in the retroviral LTR and another component that is complementary to a gRNA that is complementary to the target site in the retroviral structural gene. is there. Examples of effective combinations include GagD, LTR 1, LTR 2, LTR 3, LTR A, LTR B, LTR C, LTR D, LTR E, LTR F, LTR G; LTR H, LTR I, LTR J , LTRK, LTR L, LTR M; combinations with one of LTR N, LTR O, LTRP, LTR Q, LTR R, LTR S, or LTR T. Examples of effective combinations also include a combination of LTR3 and one of LTR-1, Gag A; Gag B; Gag C, GAG D, Pol A, or Pol B.

  The combination of LTR A and LTR B 'also resulted in the removal of a segment of the HIV-1 genome, as shown in Example 3. The compositions of the present invention are not limited to these combinations and may comprise any suitable combination of gRNAs complementary to two or more different target sites in the HIV-1 provirus.

  In certain embodiments, the target nucleic acid sequence comprises one or more nucleic acid sequences in the coding and non-coding nucleic acid sequences of the retroviral genome. The target nucleic acid sequence can be located within a sequence encoding a structural protein, a nonstructural protein, or a combination thereof. Sequences encoding structural proteins include nucleic acid sequences encoding Gag, GAG-Pol precursor, Pro (protease), reverse transcriptase (RT), integrase (In), Env, or combinations thereof It is out. A sequence encoding a nonstructural protein includes a nucleic acid sequence encoding a control protein (eg, Tat, Rev), an accessory protein (eg, Nef, Vpr, Vpu, Vif) or a combination thereof.

  In certain embodiments, the gRNA sequence comprises Gag, GAG-Pol precursor, Pro, reverse transcriptase (RT), integrase (In), Env, Tat, Rev, Nef, Vpr, Vpu, Vif or combinations thereof. Have at least 75% sequence identity to the encoding complementary target nucleic acid sequence.

  In certain embodiments, the gRNA sequence comprises Gag, GAG-Pol precursor, Pro, reverse transcriptase (RT), integrase (In), Env, Tat, Rev, Nef, Vpr, Vpu, Vif or combinations thereof. Complementary to the encoding target nucleic acid sequence.

  In other embodiments, the gRNA nucleic acid sequence has at least 75% sequence identity to a sequence comprising SEQ ID NOs: 1-57 or any combination thereof. In other embodiments, the gRNA nucleic acid sequence comprises SEQ ID NOs: 1-57.

  In another embodiment, the nucleic acid sequence comprises a sequence having at least 75% sequence identity to a sequence comprising SEQ ID NO: 1-57 or any combination thereof. In other embodiments, the nucleic acid sequence comprises the sequence defined in SEQ ID NOs: 1-57.

  In another embodiment, the composition for use in inactivating retroviral DNA integrated into the genome of a host cell to which the retrovirus is latently infected is Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). An isolated nucleic acid sequence encoding a related endonuclease and at least one guide RNA (gRNA) complementary to a target sequence in the integrated retroviral DNA, wherein the retrovirus is human immunodeficient Virus (HIV). The at least one gRNA comprises a first gRNA complementary to a target sequence in the integrated retroviral DNA; and a second gRNA complementary to another target sequence in the integrated retroviral DNA. At least including, thereby removing intervening sequences between the two gRNAs.

  In certain embodiments, the target nucleic acid sequence comprises one or more sequences in the long terminal repeat (LTR) region of human immunodeficiency virus (HIV) proviral DNA, and the structural gene and / or non-HIV-integrated DNA. One or more targets in the structural gene; or one or more targets in the second gene; or one or more targets in the first gene and one or more targets in the second gene; or in the first gene One or more targets and one or more targets in the second gene and one or more targets in the third gene; or one or more targets in the second gene and in the third or fourth gene One or more targets; or any combination thereof.

  In another embodiment, a composition for eliminating retroviruses in vitro or in vivo comprises a clustered regularly interspaced short palindromic repeat (CRISPR) related endonuclease and at least one guide RNA complementary to a target sequence in the retroviral genome ( the retrovirus is a human immunodeficiency virus (HIV). In an embodiment, the at least one gRNA comprises at least a first gRNA complementary to a target sequence in the HIV genome; and a second gRNA complementary to another target sequence in the HIV genome, thereby The intervening sequence between the two gRNAs is removed.

  In another embodiment, the composition is isolated encoding two guide RNAs (gRNAs) that are complementary to different target sequences in the clustered regularly interspaced short palindromic repeat (CRISPR) related endonuclease and retroviral genome, respectively. The nucleic acid sequence is included, and the retrovirus is human immunodeficiency virus (HIV). In embodiments, the at least one guide RNA (gRNA) comprises at least a first gRNA that is complementary to a target sequence in the HIV genome; and a second gRNA that is complementary to another target sequence in the HIV genome. This removes the intervening sequence between the two gRNAs.

  In certain embodiments, the target nucleic acid sequence comprises one or more nucleic acid sequences in the coding and non-coding nucleic acid sequences of the retroviral genome. The target nucleic acid sequence may be located within a sequence encoding a structural protein, a nonstructural protein, or a combination thereof. The above sequence encoding a structural protein is a nucleic acid sequence encoding Gag, Gag-Pol precursor, Pro (protease), reverse transcriptase (RT), integrase (In), Env or combinations thereof. Contains. The sequence encoding a nonstructural protein includes a nucleic acid sequence encoding a control protein (eg, Tat, Rev), an accessory protein (eg, Nef, Vpr, Vpu, Vif) or a combination thereof. .

  In certain embodiments, the gRNA sequence comprises Gag, GAG-Pol precursor, Pro, reverse transcriptase (RT), integrase (In), Env, Tat, Rev, Nef, Vpr, Vpu, Vif or combinations thereof. Have at least 75% sequence identity to the encoding complementary target nucleic acid sequence.

  In certain embodiments, the gRNA sequence comprises Gag, GAG-Pol precursor, Pro, reverse transcriptase (RT), integrase (In), Env, Tat, Rev, Nef, Vpr, Vpu, Vif or combinations thereof. Complementary to the encoding target nucleic acid sequence.

  In other embodiments, the gRNA nucleic acid sequence has at least 75% sequence identity to a sequence comprising SEQ ID NOs: 1-57 or any combination thereof. In other embodiments, the gRNA nucleic acid sequence comprises SEQ ID NOs: 1-57.

  Thus, the present invention also provides a method for inactivating proviral DNA integrated into the genome of a host cell in which a retrovirus is latently infected, complementary to a target site in CRISPR-related endonuclease and proviral DNA. Treating said host cell with a composition comprising a specific at least one gRNA; expressing a gene editing complex comprising said CRISPR-related endonuclease and said at least one gRNA; and inactivating proviral DNA A method including a step of converting. The gRNAs and Cas9 endonucleases listed above are preferred. In another preferred embodiment, treating the host cell in vitro or in vivo comprises treatment with at least two gRNAs, each of the at least two gRNAs complementary to a different target nucleic acid sequence in the provirus. It is. Particularly preferred is a combination of at least two gRNAs, wherein at least one gRNA is complementary to a target site in a retroviral LTR and at least one gRNA is complementary to a target site in a retroviral structural gene Contains the composition. HIV is the preferred retrovirus.

  In another embodiment, a composition for eliminating retroviruses in vitro or in vivo comprises a clustered regularly interleaved short palindromic repeat (CRISPR) related endonuclease and at least one guide RNA complementary to a target sequence in the retroviral genome ( the retrovirus is a human immunodeficiency virus (HIV), and the gRNA is a first gRNA complementary to a target sequence in the HIV genome; and It contains at least a second gRNA that is complementary to another target sequence in the HIV genome, thereby removing intervening sequences between the two gRNAs. The target nucleic acid sequence includes one or more nucleic acid sequences in the coding and non-coding nucleic acid sequences of the HIV genome. In one embodiment, the target sequence comprises one or more nucleic acid sequences in the HIV genome, wherein the one or more nucleic acid sequences encode a long terminal repeat (LTR) nucleic acid sequence, a structural protein. It includes a nucleic acid sequence, a nucleic acid sequence encoding a nonstructural protein, or a combination thereof. In certain embodiments, the nucleic acid sequence encoding the structural protein encodes Gag, Gag-Pol precursor, Pro (protease), reverse transcriptase (RT), integrase (In), Env or combinations thereof. Containing nucleic acid sequences. In embodiments, the nucleic acid sequence encoding a nonstructural protein includes a nucleic acid sequence encoding a regulatory protein, an accessory protein, or a combination thereof. Examples of regulatory proteins include Tat, Rev, or combinations thereof. Examples of accessory proteins include Nef, Vpr, Vpu, Vif or combinations thereof. In certain embodiments, the gRNA nucleic acid sequence comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NOs: 1-57. In certain embodiments, the gRNA nucleic acid sequence comprises a nucleic acid sequence comprising SEQ ID NOs: 1-57.

  In certain embodiments, the isolated nucleic acid sequence comprises at least one guide RNA (gRNA) complementary to a target nucleic acid sequence in a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -related endonuclease and retroviral genome (eg, HIV). Contains the encoding nucleic acid sequence.

  When the composition is administered as a nucleic acid or is contained within an expression vector, the CRISPR endonuclease may be encoded by the same nucleic acid or vector as the guide RNA sequence. Alternatively or additionally, the CRISPR endonuclease may be encoded in a nucleic acid that is physically separate from the gRNA sequence or in a separate vector.

(Modified nucleic acid sequence or mutated nucleic acid sequence)
In some embodiments, any of the nucleic acid sequences embodied herein can be modified or derived from a native nucleic acid sequence (eg, by mutation, deletion, substitution, nucleobase, backbone modification, etc.). . The nucleic acid sequence includes a vector, a gene editing agent, gRNA, tracrRNA, and the like. Examples of some modified nucleic acid sequences envisioned by the present invention include modified backbones (eg, phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intrasugar linkages, or short heteroatoms. Or those containing a heterocyclic intrasugar linkage). In some embodiments, modified oligonucleotides include those with a phosphorothioate backbone, and heteroatom backbones, CH ₂ --NH--O--CH ₂ , CH, --N (CH ₃ )-O --CH ₂ [known as methylene (methylimino) or MMI skeleton], CH ₂ --O--N (CH ₃ )-CH ₂ , CH ₂ --N (CH ₃ )-N (CH ₃ )-CH ₂ and O--N (CH ₃ )-CH ₂ --CH ₂ skeletons (where the native phosphodiester skeleton is represented as O--P--O--CH) Includes things to prepare. The amide skeleton disclosed by De Mesmaekeret al. Acc. Chem. Res. 1995, 28: 366-374 is also embodied herein. In some embodiments, a nucleic acid sequence having a morpholino backbone structure (Summerton and Weller, US Pat. No. 5,034,506), a peptide nucleic acid (PNA) backbone (the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, A nucleic acid sequence having a base directly or indirectly attached to the aza nitrogen atom of the polyamide backbone (Nielsen et al. Science 1991, 254, 1497). The nucleic acid sequence can also include one or more substituted sugar moieties. The nucleic acid sequence can also have sugar mimetics such as cyclobutyl instead of a pentofuranosyl group.

Nucleic acid sequences can also additionally or alternatively include nucleobase (often referred to simply as “bases” in the art) modifications and substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). . Modified nucleobases are nucleobases that are rarely or transiently found in natural nucleic acids (eg hypoxanthine, 6-methyladenine, 5-Me pyrimidine (especially 5-methylcytosine (5-methyl -2'deoxycytosine, often also referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentbiosyl HMC), and synthetic nucleobases (eg, 2-aminoadenine, 2- (methylamino) adenine, 2- (imidazolylalkyl) adenine, 2- (aminoalkylamino) adenine or other hetero-substituted alkyladenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil , 8-azaguanine, 7-deazaguanine N ₆ (6-aminohexyl) adenine and 2,6-diaminopurine)). Kornberg, A., DNA Replication, WH Freeman & Co., SanFrancisco, 1980, pp75-77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15: 4513. Also included are “ubiquitous” bases known in the art (eg, inosine). 5-Me-C substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C (Sanghvi, YS, in Crooke, ST and Lebleu, B., eds., Antisense Research) and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

  Another modification of the nucleic acid sequences of the present invention includes one or more moieties or conjugates that are chemically linked to the nucleic acid sequence to enhance the activity or cellular absorption of the oligonucleotide. Such moieties include lipid moieties (cholesterol moiety, cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid (Manoharan et al. Bioorg. Med. Chem. Let. 1994, 4, 1053)), thioesters (eg, hexyl-S-trityl thiol (Manoharan et al. Ann. NY Acad. Sci. 1992, 660, 306; Manoharan et al. Bioorg. Med. Chem. Let) 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533)), fatty chains (eg, dodecanediol or undecyl residues (Saison-Behmoaras et al. EMBO J. 1991, 10, 111; Kabanov et al. FEBS Lett. 1990, 259, 327; Svinarchuket al. Biochimie 1993, 75, 49)), phospholipids (eg di-hexadecyl-rac-glycerol or triethylammonium 1,2- Di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharanet al. Tetrahe dron Lett. 1995, 36, 3651; Shea et al. Nucl. Acids Res. 1990, 18, 3777), polyamine or polyethylene glycol chains (Manoharanet al. Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetic acid (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651), but is not limited thereto.

  It need not be uniformly modified for all positions in a given nucleic acid sequence, indeed two or more of the aforementioned modifications may be incorporated into a single nucleic acid sequence, or a single nucleoside within a nucleic acid sequence It may be incorporated inside.

In some embodiments, an RNA molecule (eg, crRNA, tracrRNA, gRNA) may be engineered to contain one or more modified nucleobases. For example, known modifications of RNA molecules include, for example, Genes VI, Chapter 9 (“Interpreting the Genetic Code”), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne. , eds. (1998, ASM Press, Washington DC). The modified RNA component, include the following: 2'-O-methyl cytidine; ^{N 4} - ^cytidine; N 4 -2'-O- dimethyl cytidine; ^{N 4} - acetyl cytidine; 5-methyl cytidine, 5 , 2'-O-dimethylcytidine;5-hydroxymethylcytidine;5-formylcytidine;2'-O-methyl-5-formylcytidine;3-methylcytidine;2-thiocytidine;lysidine;2'-O-methyl2-thiouridine;2-thio-2′-O-methyluridine;3,2′-O-dimethyluridine; 3- (3-amino-3-carboxypropyl) uridine; 4-thiouridine; ribosylthymine; 2'-O-dimethyluridine;5-methyl-2-thiouridine;5-hydroxyuridine;5-methoxyuridine; uridine 5-oxy Acid; uridine 5-oxyacetic acid methyl ester; 5-carboxymethyluridine; 5-methoxycarbonylmethyluridine; 5-methoxycarbonylmethyl-2′-O-methyluridine; 5-methoxycarbonylmethyl-2′-thiouridine; 5-carbamoylmethyluridine; 5-carbamoylmethyl-2′-O-methyluridine; 5- (carboxyhydroxymethyl) uridine; 5- (carboxyhydroxymethyl) uridine methyl ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyl-2′-O-methyl-uridine; Karboki Methylaminomethyl-2-thiouridine; dihydrouridine; dihydro ribosyl thymine; 2'-methyl-adenosine;2-methyl-adenosine; ^{N 6} N-methyl ^{adenosine; N 6,} N 6 - dimethyl ^{adenosine; N} 6, 2'-O- trimethyl 2 methylthio-N ⁶ N isopentenyl adenosine; N ^6- (cis-hydroxyisopentenyl) -adenosine; 2-methylthio-N ^6- (cis-hydroxyisopentenyl) -adenosine; N ^6- (glycinylcarbamoyl) adenosine; ^{N 6} Torre Oni carbamoyl adenosine; ^{N 6} - methyl -N ⁶ - Torre Oni carbamoyl adenosine; 2-methylthio -N ⁶ - methyl -N ⁶ - Torre Oni carbamoyl adenosine; ^{N 6} - hydroxy nor burrs carbamoyl adenosine; 2- Chiruchio -N ⁶ - hydroxy nor burrs carbamoyl adenosine; 2'-O-ribosyl adenosine (phosphate); inosine; 2 'O-methyl inosine; 1-methyl inosine; 1,2'-O- dimethyl inosine; 2' 1-methyl guanosine; N ² -methyl guanosine; N ² , N ² -dimethyl guanosine; N ² , 2′-O-dimethyl guanosine; N ² , N ² , 2′-O-trimethyl guanosine ; 2'-O-ribosyl guanosine (phosphate); 7-methyl ^{guanosine; N 2, 7-dimethyl-guanosine;} N ^2, N ^2, 7- trimethyl guanosine; Waioshin; Mechiruwaioshin; modified in the hydroxy-Wye but-thin Hydroxybutycine; peroxywitocin; queosin; epoxy quasosine; Lactosyl - Kueoshin; mannosyl - Kueoshin; 7-cyano-7-deazaguanosine; [also known as 7-formamide 7-deazaguanosine] Arakaeoshin; and 7-amino-7-deazaguanosine.

  Isolated nucleic acid molecules of the present invention can be made by standard techniques. For example, polymerase chain reaction (PCR) technology can be used to obtain an isolated nucleic acid containing the nucleotide sequence described herein. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. In general, using the sequence information at the end of the region of interest or beyond this region, oligonucleotide primers are designed that are identical or similar in sequence to the opposite strand of the template to be amplified. Various PCR strategies can also be utilized, which can introduce site-specific nucleotide sequence modifications into the template nucleic acid.

  An isolated nucleic acid may be chemically synthesized as a single nucleic acid molecule (eg, using 3 ′ to 5 ′ automated DNA synthesis using phosphoramidite methods) or chemically as a series of oligonucleotides. May be synthesized. For example, one or more long oligonucleotide pairs (eg, greater than 50-100 nucleotides) may be synthesized to contain the desired sequence. In the above synthesis, each pair of oligonucleotides contains a short complementary segment (eg, about 15 nucleotides) that forms a region that becomes double-stranded when annealed. A DNA polymerase is used to extend the oligonucleotides, resulting in one double-stranded nucleic acid molecule for each oligonucleotide pair. This double stranded nucleic acid molecule can then be ligated into a vector.

  The invention also includes a pharmaceutical composition for inactivation of an incorporated proviral HIV DNA in a mammalian subject. The composition includes an isolated nucleic acid sequence encoding a Cas endonuclease and an isolated nucleic acid sequence encoding at least one gRNA complementary to a target sequence in proviral HIV DNA. The isolated nucleic acid sequence is contained in at least one expression vector; In a preferred embodiment, the pharmaceutical composition comprises a first gRNA and a second gRNA, and as described above, the first gRNA targets a site in the HIV LTR and the second gRNA gRNA targets a site in the HIV structural gene.

  Exemplary expression vectors included in the pharmaceutical composition include plasmid vectors and lentiviral vectors, but the invention is not limited to these vectors. A wide variety of host / expression vector combinations may be used to express the nucleic acid sequences described herein. Suitable expression vectors include, but are not limited to, plasmids and viral vectors (eg, derived from bacteriophages, baculoviruses, and retroviruses). Numerous vectors and expression systems are available from companies such as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), And Invitrogen / LifeTechnologies (Carlsbad, Calif.). The marker gene can provide a selectable expression system for the host cell. For example, the marker can confer biocide resistance, such as antibiotic (eg, kanamycin, G418, bleomycin, or hygromycin) resistance. An expression vector can include a tag sequence designed to facilitate manipulation or detection (eg, purification or localization) of the expressed polypeptide. Tag sequences (such as the green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG ™ tag (Koda, New Haven, CT) sequences) typically Expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide (including insertions at either the carboxyl terminus or the amino terminus).

  The vector may also contain regulatory regions. The term “regulatory region” refers to a nucleotide sequence that affects the initiation and rate of transcription or translation and the stability and / or mobility of the transcript or translation product. The regulatory region includes promoter sequence, enhancer sequence, response element, protein recognition site, induction element, protein binding sequence, 5 ′ untranslated region and 3 ′ untranslated region (UTR), transcription initiation site, termination sequence, polyadenylation sequence , Nuclear localization signals, and introns.

  If desired, the polynucleotides of the invention may also be used with microdelivery vehicles such as cationic liposomes and adenoviral vectors. See Mannino and Gould-Fogerite, BioTechniques, 6: 682 (1988) for a description of procedures for liposome preparation, targeting and content delivery. See also Felgner and Holm, Bethesda Res. Lab. Focus, 11 (2): 21 (1989) and Maurer, R.A., Bethesda Res. Lab. Focus, 11 (2): 25 (1989).

  The compositions of the present invention result in suppression of proviral HIV-1 activation or partial or total removal of incorporated HIV-1 (Examples 2 and 3), and the present invention For example, a method of treating a mammalian subject infected with HIV) is provided. The method includes determining that the mammalian subject is infected with a retrovirus, administering an effective amount of the pharmaceutical composition as described above, and treating the mammalian subject with respect to a retroviral infection. .

  The method represents a solution to the problem of integrated provirus that is essential for the treatment and prevention of AIDS and other retroviral diseases. In the acute phase of HIV infection, HIV virions invade cells expressing unique CD4 receptor molecules. Once the virus enters the host cell, the reverse transcriptase encoded by HIV produces a proviral DNA copy of HIV RNA that is integrated into the genomic DNA of the host cell. It is this HIV provirus that is replicated by the host cell. As a result, new HIV virus particles are released that can infect other cells.

  Primary HIV infection subsides within weeks to months and usually has a long clinical “latency” period that can last up to 10 years. During this incubation period there may be no clinical symptoms and no detectable virus replication in peripheral blood mononuclear cells, and there may be little or no virus culturable in peripheral blood. However, the HIV virus continues to propagate at very low levels. In subjects treated with antiretroviral therapy, this incubation period can extend for decades or more. Antiretroviral therapy does not suppress low levels of viral genome expression, and latently infected cells (such as dormant memory T cells, monocytes, brain macrophages, microglia, astrocytes, and gut-related lymphoid cells) Is not targeted efficiently. Since the compositions of the present invention can inactivate or remove HIV provirus, treatment methods using such compositions constitute a new means of attack against HIV infection.

  The compositions of the present invention reduce or prevent new infection by retroviruses (eg, HIV) when stably expressed in latent host cells (Example 3). Accordingly, the present invention also provides a method of treatment for reducing the risk of retroviral infection (eg, HIV infection) in a mammalian subject at risk of infection. The method includes determining that the mammalian subject is at risk of HIV infection, administering an effective amount of the pharmaceutical composition as described above, and reducing the risk of HIV infection in the mammalian subject. It is out. Preferably, the pharmaceutical composition comprises a vector that provides stable and / or inducible expression of at least one of those listed above.

  The pharmaceutical composition according to the present invention may be prepared by various methods known to those skilled in the art. For example, the nucleic acids and vectors described above can be formulated in a composition for application to cells in tissue culture or a composition for administration to a patient or subject. These compositions can be prepared in a manner well known in the pharmaceutical art and are administered by a variety of routes depending on whether local or global treatment is desired and the site to be treated. obtain. Administration is by topical administration (including delivery to eye drops and mucosa (including nasal, vaginal, and rectal)), pulmonary administration (eg, inhalation or insufflation of powder or aerosol (including by nebulizer); intratracheal; Nasal, epidermal and transdermal), ophthalmic, oral or parenteral. Delivery methods to the eye include (i) local administration (instillation), (ii) injection into the subconjunctiva, periocular or intravitreal, or (iii) intraocular insertion placed in the conjunctival sac by a balloon catheter or surgery. Introduction by product. Parenteral administration includes (i) intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or (ii) intracranial administration (eg, intrathecal or intracerebroventricular) It is done. Parenteral administration may be performed in the form of a single bolus administration, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers (water based, powder based or oil based bases, thickeners, etc.) may be necessary or desirable.

  The present invention also includes pharmaceutical compositions comprising as active ingredients the nucleic acids and vectors described herein in combination with one or more pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable” (or “pharmacologically acceptable”) refers to adverse reactions, allergic reactions, or other inconveniences when administered to animals or humans as appropriate. It refers to molecular entities and compositions that do not cause any significant reactions. As used herein, the term “pharmaceutically acceptable carrier” refers to solvents, dispersion media, coatings, antibacterial agents, isotonic agents, and absorption delays that can be used as vehicles for pharmaceutically acceptable substances. Any and all of agents, buffers, excipients, binders, lubricants, gels, surfactants, and the like. In preparing the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted with an excipient, or in such a carrier (eg, capsule, tablet, sachet, Encapsulated (in the form of a paper packet or other container). Where the excipient functions as a diluent, the excipient may be a solid, semi-solid, or liquid material (eg, saline), as a vehicle, carrier, or vehicle for the active ingredient Play a role. Therefore, the composition can be a tablet, pill, powder, troche, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium), lotion, cream, It can be in the form of ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending on the intended route of administration. The resulting composition may contain additional agents (such as preservatives). In some embodiments, the carrier may be or include a lipid-based or polymeric colloid. In some embodiments, the carrier material may be a colloid formulated as a liposome, hydrogel, microparticle, nanoparticle, or block copolymer micelle. As mentioned above, the carrier material may form a capsule and the material may be a polymeric colloid.

  The nucleic acid sequences of the invention can be delivered to appropriate cells of interest. This can be achieved, for example, by using a polymer delivery vehicle, biodegradable particulate delivery vehicle, or microcapsule delivery vehicle that is optimally sized for phagocytosis by phagocytic cells (such as macrophages). For example, PLGA (poly-lacto-co-glycolide) microparticles with a diameter of about 1-10 μm can be used. Polynucleotides are stored in these microparticles, taken up by macrophages, and gradually biodegraded within the cells. This releases the polynucleotide. When the polynucleotide is released, DNA is expressed in the cell. The second type of microparticles are not directly taken up by cells, but mainly function as a sustained-release carrier for nucleic acids. The nucleic acid is taken up by cells only when released from the microparticles through biodegradation. Accordingly, these polymer particles should be large enough to prevent phagocytosis (ie, greater than 5 μm, preferably greater than 20 μm). Another way to actively take up nucleic acids is to use liposomes (prepared by standard methods). Nucleic acids may be incorporated into these delivery vehicles alone or with tissue specific antibodies. Such tissue-specific antibodies target, for example, cell types that are common latent infection carriers of HIV infection (eg, brain macrophages, microglia, astrocytes, and gut-related lymphoid cells). An antibody is mentioned. Alternatively, molecular complexes composed of plasmids or other vectors that are electrically or covalently linked to poly-L-lysine may be prepared. Poly-L-lysine binds to a ligand that can bind to the receptor of the target cell. Another means of achieving in vivo expression is to deliver “native DNA” (ie, without delivery vehicle) to an intramuscular, intradermal or subcutaneous site. In related polynucleotides (eg, expression vectors), a nucleic acid sequence encoding an isolated nucleic acid sequence comprising sequences encoding a CRISPR-related endonuclease and a guide RNA comprises a promoter or an enhancer and a promoter Is operably coupled with the combination. Promoters and enhancers are described above.

  In some embodiments, the composition of the invention has a core of nanoparticles (eg, (i) a high molecular weight linear polyethyleneimine (LPEI) complexed with DNA, and (ii) Nanoparticles covered by a shell of low molecular weight LPEI modified (PEGylated) with polyethylene glycol.

  Nucleic acids and vectors may be applied to the surface of an instrument (eg, a catheter) or contained within a pump, patch, or other drug delivery device. The nucleic acids and vectors of the invention may be administered alone or as a mixture in the presence of a pharmaceutically acceptable excipient or carrier (eg, physiological saline). Excipients or carriers are selected based on the method and route of administration. Suitable pharmaceutical carriers, along with pharmaceutical necessities for use in pharmaceutical formulations, are well-known reference books in Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopeia and the National Formulary). Have been described.

  In some embodiments, the composition can be formulated as nanoparticles encapsulating a nucleic acid encoding Cas9 or variant Cas9 and at least one gRNA sequence complementary to a target HIV, or a component thereof. A vector encoding can be included. Alternatively, the composition can be formulated as a nanoparticle encapsulating a polypeptide encoded by one or more nucleic acid compositions of the present invention.

  Regardless of whether the composition is administered as a nucleic acid or a polypeptide, the composition is formulated to promote uptake by mammalian cells. Useful vector systems and formulations are as described above. In some embodiments, the vector can deliver the composition to a particular cell type. However, the present invention is not so limited and other DNA delivery methods (eg, calcium phosphate, DEAE dextran, as well as physical delivery methods (such as electroporation, microinjection, ballistic particles, and “gene gun” systems). , Liposomes, lipoplexes, surfactants, and chemical transfection using perfluoro drug solutions).

  In other embodiments, the composition comprises cells transformed or transfected with one or more Cas / gRNA vectors. In some embodiments, the methods of the invention can be applied ex vivo. That is, the subject cells may be removed from the body, treated with the composition during culture, for example to excise the HIV viral sequence, and the treated cells returned to the subject body. The cell may be a cell of interest, or a haplotype compatible cell or cell line. The cells may be irradiated with radiation to prevent replication. In some embodiments, the cells are human leukocyte antigen (HLA) compatible cells, autograft cells, cell lines, or combinations thereof. In other embodiments, the cells may be stem cells. Examples thereof include embryonic stem cells or artificial pluripotent stem cells (artificial pluripotent stem cells (iPS cells)). Embryonic stem cells (ES cells) and artificial pluripotent stem cells (artificial pluripotent stem cells, iPS cells) have been established from a number of animal species including humans. These types of pluripotent stem cells are considered the most useful source of cells for regenerative medicine. This is because these cells can differentiate into almost any organ by appropriate differentiation induction while maintaining the ability to actively divide while retaining their pluripotency. In particular, iPS cells can be established from autologous somatic cells. Therefore, it is less likely to cause ethical and social problems compared to ES cells produced by destroying embryos. Furthermore, since iPS cells are autologous cells, it becomes possible to avoid rejection, which is the greatest obstacle in regenerative medicine or transplantation therapy.

  Isolated nucleic acid can be readily delivered to a subject by methods known in the art (eg, methods of delivering siRNA). In some aspects, Cas can be a fragment that contains the active domain of the Cas molecule, thereby reducing the size of the molecule. Therefore, Cas9 / gRNA molecules can be used clinically, similar to the approaches adopted in current gene therapy. In particular, Cas9 / double-stranded gRNA stably expressing stem cells or iPS cells for cell transplantation therapy and vaccination can be developed for use in a subject.

Transduced cells are prepared for autotransfusion according to established methods. After a culture period of about 2-4 weeks, the cells can be between 1 × 10 ⁶ and 1 × 10 ¹⁰ . In this regard, the growth characteristics of the cells vary between patients and cell types. Approximately 72 hours prior to autotransfusion of transduced cells, a portion is taken for phenotypic analysis and the percentage of cells expressing the therapeutic agent. For administration, the cells of the present invention may be administered at a rate determined by the cell type LD ₅₀ and the side effects of the cell type at various concentrations when applied to the patient's weight and overall health. Administration can be accomplished via single or divided doses. Adult stem cells may be collected using exogenously administered factors that stimulate their production and exit from tissues or voids, including but not limited to bone marrow or adipose tissue.

(Method of treatment)
In certain embodiments, the method of eliminating a retroviral genome in a cell or subject comprises a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) related endonuclease and at least one guide RNA (gRNA) complementary to a target nucleic acid sequence in the retroviral genome. Contacting a cell with or administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an isolated nucleic acid sequence encoding.

  In other embodiments, the method of inhibiting retroviral replication in a cell or subject comprises a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) related endonuclease and at least one guide RNA complementary to a target nucleic acid sequence in the retroviral genome ( contacting the cell or administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an isolated nucleic acid sequence encoding gRNA).

  In methods of treating retroviral infection (eg, HIV infection), the subject is subject to standard clinical tests (eg, through an immunoassay that detects the presence of HIV antibody or HIV polypeptide p24 in the subject's serum, or an HIV nucleic acid amplification assay). Can be identified. An amount of a composition that is given to a subject and that results in complete resolution of the symptoms of the infection, a reduction in the severity of the symptoms of the infection, or a slowing of the progression of the infection is considered a therapeutically effective amount. In addition to monitoring steps to optimize dosages and dosing schedules, the methods of the present invention may also include predicting results. In some methods of the invention, it is first determined whether the patient has a latent HIV infection and then whether to treat the patient with one or more compositions described herein. You may decide. In some embodiments, the method further comprises determining nucleic acid sequences of specific HIV possessed by the patient, and then designing guide RNAs to be complementary to those specific sequences. May be. For example, determine the nucleic acid sequence of the U3 region, R region or U5 region, or pol, gag, or env gene, region of the LTR of interest, and then one or more gRNAs to be exactly complementary to the patient sequence Can be designed or selected. The novel gRNA provided by the present invention greatly increases the chances of building an effective treatment.

  In a method for reducing the risk of HIV infection, a subject at risk of having an HIV infection is, for example, any presently sexual intercourse individual who is undergoing unprotected sexual intercourse (ie, having sex without a condom) May be a current sexual intercourse individual with another sexually transmitted disease; an intravenous drug user; or a male who is not circumcised. A subject at risk of having an HIV infection can be, for example, an individual (eg, a healthcare professional or initial responder) who will be in contact with people who are occupationally infected with HIV. Subjects at risk of having HIV infection are, for example, inmates in correctional facilities, or sex workers (ie for income-generating work or for anything other than money (such as food, drugs, or lodging)) Individual having sex).

  The present invention also provides an isolated nucleic acid sequence encoding a CRISPR-related nuclease (eg, Cas9 endonuclease) and at least one isolated gRNA encoding a gRNA complementary to a target sequence in an HIV provirus. Includes kits containing nucleic acid sequences. Alternatively, at least one isolated nucleic acid sequence can be encoded in a vector (such as an expression vector). Possible uses of the kit include treatment or prevention of HIV infection. Preferably, the kit contains instructions for use, syringes, delivery devices, buffer sterilization containers and diluents, or other reagents necessary for treatment or prevention. The kit may also contain appropriate stabilizers, carrier molecules, fragrances, and the like as appropriate for the intended use.

  While various embodiments of the invention have been described above, it should be understood that they have been presented by way of example only and not limitation. Numerous changes to the disclosed embodiments may be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Therefore, the breadth and scope of the present invention should not be limited by any of the embodiments described above.

  All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as any individual publication or patent document means. Applicants do not admit that any particular citation is "prior art" to their invention by virtue of their listing of various citations herein.

Example 1: Materials and Methods
Cloning of sgRNA in a lentiviral vector: Using the bioinformatics spCas9 sgRNA design tool for the best score of high efficiency and specificity, within the HIV-1 LTR-U3 region, about 20 sgRNA target sites, and Gag Four sgRNAs, two sgRNAs for Pol, and two sgRNAs for Env were designed (Table 1). All of these sgRNA seed sequences were cloned into a modified sgRNA expressing pKLV-Wg lentiviral vector (FIG. 1B) modified from an sgRNA lentiviral vector (Addgene # 50946). Briefly, the pKLV-Wg vector was digested with BbsI, treated with Antarctic Phosphatase, and the linearized vector was purified using the Quick Nucleotide Removal Kit (Qiagen). Equal amounts of sense and antisense guides (100 M in 1 μl) were mixed with polynucleotide kinase (PNK, 1 μl), 1 × PNK buffer and 1 mM ATP, incubated for 30 minutes at 37 ° C., and annealed in a PCR instrument. (95 ° C over 5 minutes, -1 ° C / 1 cycle 15 seconds 70 cycles). Phosphorylated oligo duplex (10 μM) was diluted 1: 100 to give a working solution (100 nM). Then 1 μl of oligo duplex (0.1 pmol), BbsI digested 3.5 μl pKLV-WG vector (0.015 pmol), 5 μl 2 × T7 ligase reaction buffer and 0.5 μl T7 DNA ligase (NEB) Mixed with. The mixture was incubated at room temperature for 15-30 minutes, chilled on ice, and transformed into Stabl3 competent cells. Pick 2-4 colonies for PCR using T351 (U6 / 5 ′) and sgRNA reverse primer. Two PCR positive clones are grown overnight at 37 ° C. in LB / Amp medium. The next day, miniprep plasmid DNA was sent to Genewiz Inc. for sequencing by T428 (hU6-sequence / 5 '/ F). The entire sgRNA expression cassette, including the U6 promoter, sgRNA and polyT terminator, was confirmed by sequence analysis using the GeneRunner program.

  EcoHIV-Luciferase Reporter Assay: HEK293T cells (5 × 10e4 / well) in 96-well plate, high glucose DMEM containing 10% FBS and antibiotics (10 U / ml penicillin and 100 μg / ml streptomycin) at 37 ° C. Cultured in a humidified environment with 5% CO2. The next day, cells were co-transfected by standard calcium phosphate precipitation using the EcoHIV-eLuc reporter vector, pLV-Cas9-RFP vector and the indicated sgRNA expressing pKLV-Wg vector. Two days after transfection, cell lysates were prepared using the ONE-Glo luciferase assay system (Promega) and luminescence was measured in a 2104 EnVision® Multilabel Reader (PerkinElmer). Data represent the mean ± SE of 4 independent transfections. Relative changes in single or paired sgRNAs compared to empty sgRNA vector controls were calculated.

  PCR genotyping, TA cloning and Sanger sequencing: HEK293T cells in 96-well plates were cotransfected with the EcoHIV-eLuc reporter vector, pLV-EF1a-spCas9-T2A-RFP vector and the indicated gRNA expression vector did. Two days later, cells were lysed with 90 μl of 50 mM NaOH for 10 minutes at 95 ° C. and neutralized with 10 μl of 1M Tris-HCl. The crude extract was used directly for PCR using Terra PCR Direct Polymerase Mix (Clontech) and the indicated PCR primers. Two cycles of standard PCR with 68 ° C. annealing / extension for 1 minute and 98 ° C. for 15 minutes were performed for 35 cycles. The products were separated on a 1.5% agarose gel. The bands of interest were gel purified and cloned into the pCRII T-A vector (Invitrogen) and the nucleotide sequence of individual clones was determined by sequencing in Genewiz using universal T7 and / or SP6 primers.

Example 2: HIV-1 gRNA screening and functional characterization for HIV-1 exclusion in vitro
To expand the range of effective gRNAs for editing the HIV proviral genome via CRISPR, search for candidate gRNAs, efficacy in suppressing HIV expression, and deletion or elimination of the HIV-1 proviral genome in host cells. Screened for inducibility.

  Candidate gRNAs specific for target sites in the HIV-1 genome were sought by bioinformatic techniques. Candidate gRNAs with minimal off-target potential (ie, potential to cause damage to multiple sites in the host genome) were selected for the highest likelihood of producing effective gene editing. A target site seed sequence for a candidate gRNA within the U3 control region of the LTR is shown in FIG. 1D. gRNA is LTR 1, LTR 2, LTR 3, LTR A, LTR B, LTR C, LTR D, LTR E, LTR F, LTR G, LTR H, LTR I, LTR J, LTR K, LTR L, LTR M , LTR N, LTR O, LTRP, LTR Q, LTR R, LTR S, and LTR T. The sequences of these gRNAs are shown in Table 1. Most of these candidate gRNAs can be paired for use with Cas9 nickase and FokI nuclease, which can reduce potential off-target effects up to 1500-fold. Candidate gRNAs targeting sites in the structural gag and pol genes are indicated by arrows in FIGS. 1C and 3A. These gRNAs include gag A, gag B, gag C, gag D; pol A, and pol B. Their sequences are shown in Table 1. Candidate gRNAs were cloned into a lentiviral reporter vector as shown in the lower panel of FIG. 1B. spCas9 was also cloned into a lentiviral reporter vector as shown in the upper panel of FIG. 1B.

  HEK293T host cells were cotransfected with an HIV reporter construct (EcoHIV-eLuc), a reporter expression construct for spCas9, and one or two reporter expression constructs for gRNA. Control cells contained a control construct ("LTRO"). Two days later, luciferase activity in the cell lysate was measured using the ONE-GLO ™ Luciferase Assay system.

  Most of the candidate LTR gRNAs given alone were effective in suppressing HIV-1 expression in host cells as determined by reduced luciferase expression (FIG. 2A). These gRNAs were then co-transfected in various combinations with gRNA targeting sites in the gag and pol genes. The combinations shown on the horizontal axis of FIGS. 2B and 2C are LTR 1, LTR 2, LTR 3, LTR A, LTR B, LTR C, LTR D, LTR E, LTR F, LTR G; LTR H, LTR I , LTR J, LTR K, LTR L, LTR M; GTR D combined with one of LTR N, LTR O, LTRP, LTR Q, LTR R, LTR S, or LTR T; and Gag A; Gag B Contained LTR 3 combined with one of Gag C, Gag D, Pol A or Pol B;

  Most of the single gRNAs (FIG. 2A) inhibited structural luciferase activity, but some enhanced or did not affect reporter activity. Data suggests that editing a single site within the LTR induces InDel mutations that can affect the binding of transcriptional activators or transcriptional repressors for HIV-1 LTR promoter activity.

  However, Gag-D gRNA paired with any of the LTR-gRNAs reduced luciferase activity by 64 to 96% (FIG. 2B). These data suggest that all designed gRNAs induced target site gene editing at the plasmid stage. The combination of Gag with any of the LTR-sgRNAs deletes the core promoter of the LTR and thus induces a significant decrease in promoter activity. The remaining reportal luciferase activity may reflect a small population of EcoHIV reporter expressing cells that do not contain either spCas9 or two sgRNAs. Note that the lowering effect requires the presence of all four plasmids: EcoHIV-eLuc, spCas9, two sgRNAs in the same cell.

  LTR-3 gRNA paired with either one of the designed Gag or Pol gRNA also dramatically reduced luciferase reporter activity by 73-93% (FIG. 2C). These data suggest that all designed gRNAs targeting the structural Gag or Pol region induced gene editing of the target site at the plasmid stage. The combination of LTR-3 with any of the sgRNAs within the structural region deletes the LTR core promoter and the structural genome between the cleavage sites, thus inducing a dramatic decrease in promoter activity. Such a proven concept is that any LTR-sgRNA and any other sgRNA that targets the structural region (eg, Gag, pol and Env) or any other non-structural region between the LTRs at both ends It is applicable to. The remaining reportal luciferase activity may reflect a small population of EcoHIV reporter expressing cells that do not contain either the spCas9 vector or the two sgRNA vectors. Note that the lowering effect requires the presence of all four plasmids: EcoHIV-eLuc, spCas9, two sgRNAs in the same cell.

  Next, it was determined whether suppression of HIV-1 expression reflects a deletion or fragment of the HIV-1 proviral genome. HEK293T cells were co-transfected with EcoHIV-eLuc reporter vector, pLV-EF1a-spCas9-T2A-RFP vector and gRNA expression vector. Two days later, cells were lysed with 50 mM NaOH for 10 minutes at 90 ° C. and neutralized with 1 M Tris-HCl. The crude extract was used directly for PCR using Terra PCR Direct Polymerase Mix (Clontech) and the indicated PCR primers.

  When using the first primer set (FIG. 3A), after cleavage of the designed target site, the PCR fragment showed the expected size in 17 out of 20 LTR sgRNAs. Only LTR-F, LTR-G and LTR-K show no exclusion, suggesting that they cannot cleave 5'-LTR or are less effective at cleaving 5'-LTR. This is consistent with ineffective or less effective results using single sgRNA transfection of LTR-F, LTR-G and LTR-K, as shown in FIG. 2A. Among the effective sgRNAs, the cleavage efficiency as detected by the ratio of the cleaved fragment to the corresponding uncleaved fragment was highest in the LTR sgRNA. Interestingly, an additional band with a size exceeding the expected cleavage fragment was observed in most pairs.

  When using the second primer set (FIG. 3B), two weak bands indicating non-specific PCR products were observed in all samples. However, after cleavage of the designed target site, the unique PCR fragment showed the expected size in 16 of the 20 LTR sgRNAs. Among the effective sgRNAs, the cleavage efficiency as detected by the expected PCR fragment intensity was highest in LTR-Q, L, B, S, O, C, I sgRNA.

  Next, further combinations of sgRNA targeting the LTR and various structural genes were tested. The combination is shown in FIG. 3C. Among LTR-1, 2, 3 sgRNA, LTR-1 showed the highest efficiency. Gag-A, C, D and Pol-A, B showed strong exclusion efficiency. Gag-B showed no cleavage (right panel confirmed repeatedly by PCR). Furthermore, an additional band of a size exceeding the expected cleavage fragment was observed in the pair for 5 'LTR cleavage.

  It was found that gRNA pairs complementary to the target site in the LTR U3 region also resulted in a deletion in the HIV-1 proviral genome. Sample preparation and Direct PCR were performed as previously described. The cut PCR fragment was extracted for TA cloning and Sanger sequencing (FIG. 4). Representative TA cloning and Sanger sequencing supported a 296 bp deletion between LTR-1 and LTR-3 and an additional 180 bp insertion between the two cleavage sites. Sequencing of 15 clones showed a similar pattern of complete ligation without any indel mutation between the two cleavage sites. The additional insert of 180 bp matches the vector sequence by NCBI Blast.

  In summary, the results show that most of the candidate gRNAs eliminate the predicted HIV-1 genomic sequence between the two target sites and suppress proviral expression, as shown by luciferase reporter activity. Show. In particular, the combination of viral structural gRNAs with one or two LTR gRNAs resulted in higher efficiency genomic exclusion. These experimental results expand the range of gRNAs that can be employed in the CRISPR system to cause efficient cleavage of the HIV-1 genome.

Example 3: Novel gRNA combination and vector system for elimination of integrated HIV-1 genome from latently infected T cells using CRISPR / Cas9 gene editing system
Experiments were performed to further expand the range of effective gRNAs against HIV-1 and to increase the freedom of delivery of gRNA and Cas9 to host cells.

  First, the strategy of combination processing was examined. A novel combinatorial treatment of LTR B 'gRNA with LTR A described previously in Hu, et al, 2014 was employed. The sequences of LTR A and LTR 'are shown in FIG. 11A, and their positions in the HIV-1 LTR are shown in FIGS.

  Combined expression of Cas9, LTR A and LTR B ′ gRNA suppresses the activation of latent HIV provirus and causes excision of the proviral sequence: the experiment illustrated in FIG. 6A was performed using the Jurkat2D10 reporter T cell Conducted in stocks. This cell line contains an HIV-1 provirus that is elicited for transcription and has eGFP as a reporter of proviral activation instead of Nef. Activation is induced with phorbol 12-myristate 13-acetate (PMA) or trichostatin A (TSA). The integrated HIV-1 reporter sequence is shown in FIG. 6A. Fluorescence micrographs of induced 2D10 cells (right panel) and uninduced 2D10 cells (left panel) are shown in FIG. 6B. Flow cytometric analysis of induced 2D10 cells (right panel) and uninduced 2D10 cells (left panel) is shown in FIG. 6C.

2D10 reporter cells (2 × 10 ⁶ / condition) were electroporated with 10 μg of control pX260 plasmid or 5 μg each of pX260 LTR-A and pX260 LTR-B ′ plasmids (NeonSystem, Invitrogen, 10 ms / 1350 V pulse, 3 times ). After 48 hours, the medium was replaced with medium containing 0.5 ug / ml puromycin. One week after selection, puromycin was removed and allowed to grow for another week. The cells also expressed Cas9 with a FLAG tag.

  The cells were then diluted to a concentration of 10 cells / ml and seeded in 96 well plates at 50 ul / well. Two weeks later, single cell clones were screened for GFP-tagged HIV-1 reporter reactivation (12 hours PMA 25 nM / TSA 250 nM treatment) using a flow cytometer.

  Clones expressing Cas9, LTR A and LTR B 'were compared to clones expressing only Cas9 (Figures 7A-7C). Only clones expressing Cas9, LTR A and LTR B 'were found to fail to show HIV-1 reporter reactivation after PMA induction (Figure 7B, lower panel). On the other hand, clones expressing only Cas9 showed HIV-1 reporter reactivation as evidenced by EGFP expression (FIG. 7B, upper panel).

  It was next determined whether suppression of reactivation of the latent reporter HIV-1 provirus contributed to successful excision of the proviral sequence from the host genome. The DNA obtained from the clones analyzed in the previous experiment was subjected to PCR to amplify the provirus env gene sequence motif RRE or the genomic sequence adjacent to the integrated reporter provirus (MSBR1 gene). The T position of the primer is shown in FIG. 8A.

  PCR analysis shows that clones expressing the combination of Cas9 and LTR A / B ′ cannot show PCR products (including RRE and MSRB1) (representing excision of DNA containing these sequences). Indicated. On the other hand, RRE and MSRB1 were amplified in clones expressing only Cas9 and were readily detectable (FIG. 8B). Long distance PCR genotyping confirmed that expression of the LTR A / B combination resulted in a 652 bp excision extending between the 5'U3 and 3'U3 regions (FIG. 8C). The excision was confirmed by sequencing the truncated lariat from the integration locus on chromosome 16 (FIG. 8D). Taken together, the above results confirm that suppression of HIV-1 provirus reactivation by LTR A / B 'and Cas9 expression was caused by excision of the proviral sequence from the host genome.

  It was also observed that stable expression of LTR A / B 'and Cas9 protected 2D10 from new infection by HIV-1. Clones were characterized for Cas9 expression by Western blotting (FIG. 9B) and LTR B ′ expression by reverse PCR (FIG. 9C). The clone was infected with HIV-1 NL4-3-EGFP-P2A-NEF reporter virus, and the progress of infection was monitored by FACS analysis. Only clones expressing both LTR A / B 'and Cas9 were resistant to HIV-1 infection. This is shown by dramatically lower levels of EGFP for clones lacking either LTR A / B '(ctrl7) or Cas9 (AB8) (Figure 9A).

  Delivery of Cas9 / gRNA by lentivirus allows time-controlled and efficient targeting of proviral sequences: can lentiviral vectors then be used for expression of Cas9 / gRNA components in host cells? Decided whether or not. Lentiviral vectors provide a versatile means of adaptive expression and various drugs are available that can induce lentiviral vectors. Lentivirus expressing RFP-Cas9 (red fluorescence) and / or LTR A / B ′ gRNA (BFP marker, blue fluorescence) was introduced into Jurkat2D10 at MOI5 (FIG. 10A). After 72 hours, GFP reporter virus was reactivated using PMA / TSA treatment and quantified by flow cytometry. A dot plot analysis is shown in FIG. 10B. HIV reactivation can be detected as an upward (green) shift in the induced dot plot (eg, upper left panel vs. lower left panel in FIG. 10B). It can be seen that only cells into which both Cas9 and LTR A / B 'have been introduced show a meaningful cell fraction that does not show a green upshift (Figure 10B, lower right panel). The results confirm that Cas9 / gRNA can be efficiently delivered by lentiviral vectors.

  Cas9 / LTR A / B ′ expression does not show detectable off-target effects and causes minimal change in adjacent gene expression: efficient excision of HIV-1 provirus by CRISPR editing is similar to the target sequence Usefulness is reduced when accompanied by induced mutations in the normal host genome containing the sequences to Six predicted / possible off-target sites for LTR A / B 'were examined in Jurkat clones in which the HIV-1 genome was successfully eliminated. The sequences of LTRA and LTRB 'are shown in Figure 11A. Indel mutations were not observed by either the Surveyor assay reaction (FIG. 11B) or Sanger sequencing. The location of the second exon of the MSRB1 gene on chromosome 16 and the HIV-1 reporter integration site in the adjacent gene is shown in FIG. 11D. The level of expression of the gene adjacent to the integration site after exclusion of the HIV-1 sequence was measured by qRT-PCR and compared to the level of expression in control cells. The results in FIG. 11E show that Cas9 and LTR A / B 'do not significantly affect the expression of genes adjacent to the excised HIV sequence.

Example 4: Functional screening of guide RNA for AIDS healing targeting regulatory and structural HIV-1 viral genomes
In this study, the best gRNA targeting the HIV-1 LTR and viral structural regions were identified to optimize gRNA pairing that could efficiently eliminate the HIV-1 genome.

  Highly specific gRNAs were designed using bioinformatics tools to demonstrate their ability to induce Cas9 to cleave HIV-1 proviral DNA, high-throughput HIV-1 luciferase reporter assay and fast Direct-PCR. Evaluated using genotyping.

〔result〕
(Bioinformatics screening of sgRNA with high efficiency and low off-target effect)
The efficiency and specificity of the target RNA is an important concept for the use of Cas9 / gRNA in infectious diseases. Various computational programs have been developed for the design and selection of gRNA for spCas9-gRNA systems where a 20 bp seed sequence and NRG PAM are used. Most of the gRNA design programs have been developed to predict off-target effects, and very few programs can predict cleavage efficiency. Twenty gRNAs targeting HIV-1 with high score cleavage efficiency and cleavage specificity for the human genome were designed using the following criteria. (1) Targeting the -18 to -418 bp of the LTR-U3 promoter to disrupt early transcription of HIV-1 (suppressing viral production), this 400 bp region is eliminated in most LVs, and thus LV Avoids self-cleavage; (2) avoids transcription factor binding sites that may affect host cell gene expression due to high homology; (3) enables the elimination of the entire proviral DNA between LTRs To match both single LTRs to: (4) an off-target score not exceeding 50%; and (5) applicability of double spCas9 nickase or RNA-induced dimeric FokI nuclease. A small number of gRNAs targeting the structural regions Gag and Pol (FIG. 1C) with the expectation of obtaining the 9 best combinations of gRNAs to eliminate the entire HIV-1 genome. The Env structural region was not selected due to less conservation in this structural sequence among different strains. The LV gene delivery system was selected separately to express spCas9 and gRNA for the following reasons. (1) LV itself provides many benefits for highly efficient gene therapy in difficult to transfect HIV latent cell lines, animal experiments and potential clinical applications (LV without integration; Hu P, et al. Mol Ther Methods Clin Dev 2015, 2: 15025; Liu KC, et al. Curr Gene Ther 2014, 14: 352-364); (2) Separate spCas9 LVs ensure good package efficiency for large sized spCas9 genes; (3) LVs that express separate gRNAs can be used to clone multiple gRNA expression cassettes into one vector for good packaging efficiency.

(Functional screening in HEK293T cells to identify efficient gRNA)
For functional, rapid screening of the best targets, EcoHIV-eLuc reporter assays were performed using a fast processing Envision multiplate reader. (1) Contain all components necessary for HIV-1 replication except HIV Env, (2) Convenience that can be manipulated in safety level II vessels for biological research due to Env deletion (3) Because bioluminescence is more sensitive than fluorescence and the eLuc reporter can be used to detect less than 10 single cells (Song J, etal. J Gen Virol 2015,96: 3131-3142) The EcoHIV-eLuc reporter was selected. The HEK293T cell line was selected for highly efficient transfection using a cost-effective calcium phosphate precipitation method. According to a single gRNA transfection, most of the gRNAs targeting the LTR promoter and structural regions can only result in minimal reduction in EcoHIV proviral reporter production, while some enhance promoter activity, Or it was found that there was no effect (FIGS. 2A, 2B). The enhancement of promoter activity is consistent with recent reports that spCas9 / gRNA-induced DSBs within the promoter of neuronal early response genes stimulate their expression (Madabhushi R, et al. Cell 2015, 161: 1592-1605). A single gRNA was postulated to induce unique cleavage of the target site, resulting in an InDel mutation in the target region and / or a deletion of the entire proviral DNA between the LTRs at both ends. Mutations in the promoter can affect the functional activity of transcriptional activators and / or repressors that can lead to enhanced or reduced transcriptional activity. Mutations in the structural region can result in a shift in the open reading frame of the HIV-1 structural protein, thus reducing eLuc reporter expression.

  In order to achieve a more reliable and sensitive screening of functionally efficient cRNAs, each pair of gRNAs (each LTR gRNA for one of a plurality of gRNAs targeting a structural region) Co-transfected. According to this strategy, a more dramatic drop in reporter virus was observed due to the deletion of a large fragment between the 5 'or 3' LTR and the structural region. As in the example shown in FIG. 2B, all combinations of any one of GagD and LTR-gRNA were stronger and significantly reduced eLuc expression than using a single gRNA. Half of LTR-gRNA (10/20) showed a 90% reduction in eLuc activity. Similarly, as in other examples shown in FIG. 2C, LTR-R gRNA paired with any one of GagA-D or PolA-B significantly reduces luciferase reporter activity by 7-23% I let you. In addition, selection of GagD or LTR-R for pairing will result in HIV-1 lacking part of the Gag sequence and all of the Pol sequence, as well as those PAM sites available for the Cas9 system of Staphylococcus aureus. Available for latent cell lines (Jadlowsky JK, et al. Mol Cell Biol 2014, 34: 1911-192) and Tg26 transgenic mice (Kopp JB, et al. Proc Natl Acad Sci USA 1992, 89: 1577-1581) Based on their target sites. These data indicate that the combination of LTR-gRNA with structural gRNA is better and makes it easier to strategy efficient gRNA screening using the high-throughput HIV-1 eLuc reporter assay. Showed evidence. All designed gRNAs are functional to reduce the expression of the EcoHIV-eLuc reporter, consistent with a high efficiency prediction score from bioinformatics analysis.

(Efficient gRNA identification using Direct-PCR genotyping)
To ascertain whether these candidate gRNAs are functional in cleaving the appropriate target as designed, genotyping analysis was performed with a pair of gRNAs as shown (Figure 3D) and This was done using a DNA sample with the corresponding PCR primer. The Direct-PCR approach does not require DNA extraction and purification and is therefore more convenient for genotyping screening. When one of the gRNAs targeting the structural region is used to pair with the LTR target site, PCR genotyping remains after deletion of the fragment between 5 'LTR and Gag (residual ) Clearly resulted in a new fragment (denoted as a deletion for convenience of description) derived from the viral LTR and Gag sequences. Consistent with the eLuc reporter assay, almost all gRNAs in varying degrees of wild-type band that can be easily amplified by standard PCR conditions for 5'-LTR / Gag due to size (1.3 kb) A decrease was induced. After cleavage, the various degrees of deletion shown were detected in most combinations (Figure 3E). Interestingly, more fragments (denoted as insertions), more than the expected deletions, were observed in most combinations for 5′-LTR-Gag cleavage (FIG. 3E). Quantification of the intensity of the wild-type band showed that LTR-O had the highest efficiency (followed by I, C, A as shown in the box) (FIG. 3E). This cleavage efficiency pattern of the wild type band is because amplification of PCR products in the mixture population generally favors smaller size products. It did not completely correlate with the decrease pattern of eLuc reporter activity (FIG. 2B). On the other hand, a weak reduction of the wild-type band in some pairs can cause various degrees of small InDel mutations within the gRNA target site without any fragment deletion or insertion. To avoid the strong impact of PCR preferential amplification, PCR genotyping expected to produce a 7 kb wild-type PCR product that should not be amplified by the PCR settings used was determined by 3′-LTR and Gag This was carried out with primers directed to (Figure 3E, 3I, 3J). Among the gRNAs detected by a single PCR product, the fragment deletion pattern (FIGS. 3E, 3I, 3J) was consistent with the pattern revealed by relative ratio changes (FIG. 3E). The LTRK and Gag pair showed no deletion or insertion fragment bands in all four sets of PCR genotyping reactions (FIGS. 3E, 3F, 3I, 3J), corresponding to only a 7% reduction of the wild type band. (FIG. 3E). The LTR-F vs. GagD pair showed a weak deletion band in one set of PCR reactions (31), corresponding to a 17% reduction of the wild type band (FIG. 3E). The LTR-G, P to GagD pair showed about 50% reduction of the wild-type band, resulting in cleavage of the 5 ′ LTR-Gag (FIG. 3F) or Gag-3 ′ LTR (FIGS. 3F, 3I, 3J) . PCR Lenotyping using LTR-R to pair with any one of GagA-D and PolA-B and the corresponding primers shown also predicted the new fragment (deficient (FIG. 3G, 3H) and additional insertions to 5′-LTR / Gag (FIG. 3G) to varying degrees in all gRNAs tested except Gag-B gRNA, with very weak editing ability (FIG. 3G). However, all these combinations with weak or no deletion genotypes still showed a dramatic reduction in EcoHIV-eLuc reporter activity (FIG. 2B). This may not be due to two gRNA plasmids transfected into the same cell where a single gRNA is still very effective at inducing small InDel mutations, or may be due to only one of the gRNA plasmids. Taken together, these data demonstrate that Direct-PCR genotyping provides a reliable and fast tool to confirm the presence of fragmented deletions and / or insertions. However, evaluation of effective HIV-1 exclusion by various gRNAs requires a combination of functional reduction by viral reporter assay and proviral DNA fragment excision by PCR genotyping for 5'-LTR or 3'-LTR And

(Confirmation of fragment insertion / deletion mutation by TA cloning and Sanger sequencing)
In order to confirm the cleavage efficiency of spCas9 / gRNA and to examine the pattern of deletion / insertion mutations after cleavage, three representative samples of PCR cloning and PCR genotyping for Sanger sequencing were selected. LTR-R / GagA paired expression resulted in the deletion of a 519 bp fragment between the LTR-R and GagA target sites (FIG. 12A). Co-expression of LTR-L / GagD (FIG. 12B) and LTR-M / GagD (FIG. 12C) resulted in the deletion of a 772 bp or 763 bp fragment between each pair of target sites. further. They caused various degrees or types of small InDel. In some cases, large insertions or additional sequences (eg, 159-359 bp) were identified (FIGS. 3E, 12B, 12C). NCBI Blast analysis showed that these additional sequences were derived from an exogenous vector rather than an endogenous host cell gene. These results indicate that most of these candidate gRNAs can efficiently mediate targeted disruption of the HIV genome that has been integrated either by excision or insertion / deletion.

[Discussion]
A gRNA complex that provides a reliable improvement to assess the DNA cleavage efficiency of Cas9 / gRNA can induce large fragment deletions between target sites (Hu W, et al. Proc Natl Acad Sci USA). 2014,111: 11461-11466). In this study, this proven concept was further confirmed by screening various complexes of 26 gRNAs. Most of the designed gRNAs are very effective in eliminating the expected HIV-1 genomic sequence between the two selected target sites (leading to significant excision of HIV-1 reporter virus) But proved. In particular, the combination of viral structural gRNAs with one or two LTR gRNAs resulted in a very efficient genome exclusion and an easier approach using Direct-PCR and high-throughput reporter screening. The efficacy and specificity of the gRNAs selected in this study to excise HIV-1 proviral DNA are pre-clinical animal studies and clinical patients using the Cas9 / gRNA approach for the following reasons: Promising success in trials. (1) These gRNAs can serve as a readily available selection source for developing viral and non-viral gene therapy; (2) For individual HIV patients, these gRNAs have high HIV mutation rates. Nevertheless, it can be used as a master to screen new gRNAs specifically designed for any HIV isolate; (3) easy gRNA cloning, fast reporter screening and reliable Direct-PCR Genotyping provides feasibility for the actual use of Cas9 / gRNA in individual medicines.

  Not all designed gRNAs exhibit the activity necessary to cleave the expected cleavage site. Several approaches have been developed to further evaluate the efficiency of genomic variants induced by the Cas9 / gRNA approach. Continuous improvement of computer programs for predicting efficiency has been attempted using the host cell genome as a design target (Doench JG, et al. Nat Biotechnol 2014, 32: 1262-1267; Gagnon JA, et al PLoS One 2014,9: e98186; LiuH, et al. Bioinformatics 2015) may not be reliable for use against exogenous genomes (eg, infectious viruses). Sanger sequencing of target regions via PCR cloning provides high sensitivity and specificity for determining genome editing efficiency (SanderJD, et al. Nat Biotechnol 2011, 29: 697-698), but for high-throughput screening Excessive labor. Surveyor assay based on mismatch (Qiu P, et al. Biotechniques 2004, 36: 702-707; Kim JM, et al. Nat Commun 2014, 5: 3157; Dahlem TJ, et al. PLoS Genet 2012, 8: e1002861) and high Resolution melt assay (Bassett AR, Liu JL. J Genet Genomics 2014, 41: 7-19) is sensitive to detect small InDel mutations, but poor specificity tends to produce false positive results It is in. Restriction fragment length polymorphism (RFLP) assays require the presence of a restriction enzyme site, with a small target region in most cases (Kim JM, et al. Nat Commun 2014, 5: 3157) . Next generation sequencing provides a reliable and specific approach, but is expensive and time consuming (Guell M, et al. Bioinformatics 2014, 30: 2968-2970). In recent years, PCR-based assays have provided a reliable and easy way to quantify editing efficiency, but they require robust primer design, microdegradation or capillary sequencers (Brinkman EK, et al. Nucleic Acids Res 2014,42: e168; Carrington B, etal. Nucleic Acids Res 2015; Yu C, et al. PLoS One 2014,9: e98282). Here, a cost-effective and reliable high-speed screening platform identifies effective gRNA using a very sensitive, high-throughput bioluminescent reporter assay with fast Direct-PCR genotyping In order to be established. The reporter assay relies on the elimination of large fragments between two gRNA target sites and a small InDel mutation at each gRNA site. Fragmental elimination can result in loss of promoter activity or reporter expression, and InDel mutations can alter promoter control or induce a shift in the open read frame of the viral protein. All these phenomena subsequently affect the activity of the reporter. PCR genotyping relies on fragmented cleavage and efficient religation between the remaining terminal DNA. The presence of the religated PCR fragment shows good evidence for the effectiveness of both gRNAs. Religation efficiency is dependent on cell division and thus PCR genotyping can be limited in the case of non-dividing cells. In addition, the PCR conditions for some primers require optimization to achieve the best efficiency of genotype and determination.

  The purpose of this study is to screen and identify effective gRNAs by establishing reliable and sensitive high-throughput assays. EcoHIV − in HEK293T cells because a small amount of reporter plasmid (1:20) against the spCas9 / gRNA component can guarantee target cleavage in all of the reporter expressing cells and maximize the detection efficiency of luciferase reporter assays and PCR genotyping. Transient transfection of the eLuc reporter was chosen as the test platform. On the other hand, stable EcoHIV-eLuc cell lines based on HEK293T cells (FIGS. 13A-13E, 14A-14D), which are closer to the actual situation of HIV-1 latency, are poorly detected in both luciferase reporter assays and PCR genotyping Sensitivity was shown. This is because even though the transfection efficiency is close to 80-90%, EcoHIV-eLuc expressing cells without gRNA plasmid are always present after transfection and thus the eLuc reporter is steadily expressed. Additional advantages of transient reporter transfection include cost-effective, easy-to-read transfection and fast-processing luminescence measurements. Importantly, the identified gRNA is effective in cells or cell lines that are actually latently infected with HIV and can be further used in animal testing and clinical applications. Transiently transfected EcoHIV-eLuc reporter (episomal DNA) does not reflect occult HIV proviral DNA in the host genome (nucleus), but gene editing mediated by spCas9 / gRNA is (Hu W, et al. Proc Natl Acad Sci USA 2014, 111: 11461-11466) and other viruses (Ramanan V, et al. Sci Rep 2015, 5: 10833; Yuen KS, et al. J Gen Virol 2015, 96: 626-636) function in similar efficiency between episomal and nuclear DNA. Furthermore, in addition to the integrated HIV-proviral DNA, efficient cleavage of episomal DNA has been demonstrated by HIV (Hu W, et al. Proc Natl Acad Sci USA 2014, 111: 11461-11466) and other infectivity. Allows new preventive measures against new infections of the virus (Peng C, Lu M, Yang D. Virol Sin 2015, 30: 317-325).

  Several perturbing factors can affect transient transfection efficiency and transgene expression for comparative analysis of various gRNAs. Several precautions were taken to minimize this. 1) A master mixture of reporter plasmid and spCas9 plasmid was prepared to ensure equal amounts of these plasmids shared in each group of gRNA; 2) Renilla luciferase reporter (1: 100) was prepared for transfection efficiency. Used for normalization; 3) Large scale transfections were performed in 96-well plates in 4-6 sets simultaneously for all gRNAs; and 4) All data were empty gRNA controls in each experiment. And expressed as a relative change.

  One gRNA targeting the LTR region can remove the entire proviral DNA due to cleavage of the LTR at both ends, but the elimination efficiency was not clear as shown by the EcoHIV-eLuc reporter assay. It is also possible because HIV-1 can not distinguish 5'-LTR from 3'-LTR after standard PCR with primers directed to LTR after deletion of the fragment between the two LTR target sites. A long range of PCR is required to confirm the elimination of the entire proviral DNA (Hu W, et al. Proc Natl Acad Sci USA 2014, 111: 11461-11466). Two gRNAs targeting the LTR region induced fragmentary cleavage within each LTR region. The cleavage suppresses LTR promoter activity and reduces the stability of HIV-1 RNA, thus improving overall clearance efficiency as we have previously demonstrated (Hu W, et al. 2014). . In this study, new evidence of the principle was examined that any pair of gRNAs between the LTR region and the structural region would provide a better approach to assess HIV-1 exclusion efficiency. This method results in a dramatic functional decline in HIV-1 reporter virus production resulting in three cleavages, 5′LTR + Gag, Gag + 3′LTR, and LTRs at both ends, facilitated by a sensitive and fast-processing bioluminescent reporter assay Can be monitored. These cleavages can be detected efficiently and reliably by standard and fast Direct-PCR genotyping using primers directed to the LTR and structural regions. Similarly, a cocktail of two LTR gRNAs plus one or more structural gRNAs may provide an optimal and economic improvement to eliminate the HIV-1 genome in preclinical and clinical settings.

  The potential for off-target effects on the Cas9 / gRNA approach is a major concern in the field of genome editing. Stringent gRNA design, functional screening and modification of the Cas9 approach have been developed to improve the specificity of genome editing. A very rare example of off-target effects on spCas9 / gRNA in cultured cells has been confirmed by whole genome sequencing (WGS) (Hu W, et al. 2014; Zuckermann M et al. Nat Commun 2015, 6: 7391 SmithC, et al. Cell Stem Cell 2014, 15: 12-13; Veres A, et al. Cell Stem Cell 2014, 15: 27-30; Yang L, et al. Nat Commun 2014, 5: 5507). A newly developed unbiased profiling approach enables high specificity of the spCas9-gRNA system (Ran FA, et al. Nature 2015, 520: 186-191; Tsai SQ, et al. NatBiotechnol 2015, 33 : 187-197; Frock RL, et al. Nat Biotechnol 2015, 33: 179-186). In vivo off-target is expected to be very few due to epigenetic protection. In this study, exogenous viral DNA was analyzed against the host genome for the best score of efficiency and properties. No cytotoxicity was observed during gRNA screening. Dual spCas9 nickase and RNA-induced FokI nuclease have been shown to reduce potential off-target effects up to 1500-fold (Ran FA, et al. Cell 2013,154: 1380-1389; Mali P, et al. NatBiotechnol 2013, 31: 833-838; Wyvekens N, et al. Hum Gene Ther 2015, 26: 425-431; Tsai SQ, et al. Nat Biotechnol 2014, 32: 569-576).

  In conclusion, most of the designed gRNAs efficiently exclude the expected HIV-1 genomic sequence between the two selected target sites and affect eLuc reporter activity. In particular, the combination of viral structural gRNA with one or two LTR gRNAs has led to an easier approach to highly efficient genomic exclusion and PCR genotyping. Screening using the HIV-1 eLuc reporter assay and Direct-PCR genotyping provides a reliable, fast and convenient approach to screen for effective HIV-1 gRNA. This can be utilized for high-throughput gRNA library screening for any new HIV-1 isolates and other infectious viruses in a new era of individual / precision medicine.

  It will be understood that the present invention has been described by way of example, and that the terminology used is intended to be in the nature of the words for purposes of explanation and not limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims (49)

A composition for use in inactivating retroviral DNA integrated into the genome of a host cell to which the retrovirus is latently infected, comprising:
Includes isolated nucleic acids encoding Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) related endonucleases and at least one guide RNA (gRNA) complementary to the target sequence in the integrated retroviral DNA ,
A composition wherein the retrovirus is human immunodeficiency virus (HIV).   A first gRNA wherein the at least one gRNA is complementary to a target sequence in the integrated retroviral DNA; and a second gRNA complementary to another target sequence in the integrated retroviral DNA. 2. The composition of claim 1, comprising at least, thereby removing intervening sequences between the two gRNAs.   The composition of claim 2, wherein the target nucleic acid sequence comprises one or more nucleic acid sequences in coding and non-coding nucleic acid sequences of the HIV genome.   The target sequence includes one or more nucleic acid sequences in HIV, and the one or more nucleic acid sequences include a long terminal repeat (LTR) nucleic acid sequence, a nucleic acid sequence encoding a structural protein, and a nonstructural protein. 4. The composition of claim 3, comprising the encoding nucleic acid sequence, or a combination thereof.   A nucleic acid sequence wherein the sequence encoding a structural protein encodes Gag, Gag-Pol precursor, Pro (protease), reverse transcriptase (RT), integrase (In), Env or a combination thereof. 5. A composition according to claim 4 comprising.   5. The composition of claim 4, wherein the sequence encoding a nonstructural protein comprises a nucleic acid sequence encoding a control protein, an accessory protein, or a combination thereof.   7. The composition of claim 6, wherein the control protein comprises Tat, Rev, or a combination thereof.   7. The composition of claim 6, wherein the accessory protein comprises Nef, Vpr, Vpu, Vif or combinations thereof. The target nucleic acid sequence is one or more sequences in the long terminal repeat (LTR) region of human immunodeficiency virus (HIV) proviral DNA and one in the structural and / or nonstructural genes of the DNA into which HIV is integrated Or more targets; or one or more targets in the second gene; or one or more targets in the first gene and one or more targets in the second gene; or one or more targets in the first gene And one or more targets in the second gene and one or more targets in the third gene; one or more targets in the second gene and one or more targets in the third or fourth gene; 4. The composition of claim 3, comprising or any combination thereof.   4. The composition of claim 3, wherein the gRNA nucleic acid sequence comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NOs: 1-57.   11. The composition of claim 10, wherein the gRNA nucleic acid sequence comprises a nucleic acid sequence comprising SEQ ID NOs: 1-57. A method of inactivating integrated retroviral DNA in a mammalian cell, comprising:
Complementary to at least one isolated nucleic acid sequence encoding a gene editing complex containing a clustered regularly interspaced short palindromic repeat (CRISPR) -related endonuclease and the target sequence in the integrated retroviral DNA Exposing the cell to a composition comprising at least one guide RNA (gRNA); expressing the gene editing complex; and inactivating the integrated retroviral DNA. ,Method.   The integrated retroviral DNA is human immunodeficiency virus (HIV) DNA, and the at least one isolated nucleic acid sequence is complementary to the target nucleic acid sequence in the LTR region of the HIV DNA. The method of claim 12, wherein the method encodes a first gRNA complementary to a second gRNA complementary to a target nucleic acid sequence in the structural gene of the HIV DNA.   13. The method of claim 12, wherein the gRNA nucleic acid sequence comprises a nucleic acid sequence having at least 75% sequence identity to a nucleic acid sequence comprising SEQ ID NOs: 1-57.   The method of claim 14, wherein the gRNA nucleic acid sequence comprises a nucleic acid sequence comprising SEQ ID NOs: 1-57. A pharmaceutical composition for inactivation in mammalian subjects of integrated retroviral DNA comprising:
An isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) related endonuclease; and encoding at least one guide RNA (gRNA) complementary to a target sequence in retroviral DNA; A pharmaceutical composition comprising at least one isolated nucleic acid sequence, each of said isolated nucleic acid sequences being contained in at least one expression vector.   The integrated retroviral DNA is human immunodeficiency virus (HIV) DNA and the at least one gRNA is complementary to a first target sequence in the HIV DNA and in the HIV DNA. 17. A pharmaceutical composition according to claim 16, comprising a second gRNA complementary to the second target sequence.   The pharmaceutical composition according to claim 16, wherein the first target sequence is located in the LTR of the HIV DNA and the second target sequence is located in the structural gene of the HIV DNA.   17. The pharmaceutical composition of claim 16, wherein the gRNA nucleic acid comprises a sequence having at least 75% sequence identity to a nucleic acid sequence comprising SEQ ID NOs: 1-57.   20. A pharmaceutical composition according to claim 19, wherein the gRNA sequence comprises a nucleic acid sequence comprising SEQ ID NOs: 1-57. A method of treating a mammalian subject infected with human immunodeficiency virus (HIV) comprising:
17. A method comprising: determining that a mammalian subject is infected with HIV; administering an effective amount of the pharmaceutical composition of claim 16; and treating the mammalian subject for HIV infection. . A method of treatment for reducing the risk of human immunodeficiency virus (HIV) infection in a mammalian subject at risk of infection comprising:
Determining that the mammalian subject is at risk of infection; administering an effective amount of the pharmaceutical composition of claim 16; and reducing the risk of HIV infection in said mammalian subject. Is that way. A kit for the treatment or prevention of human immunodeficiency virus (HIV) infection comprising:
At least one isolated nucleic acid sequence encoding a CRISPR-related endonuclease and at least one nucleic acid sequence encoding one or more guide RNAs (gRNA) each complementary to a target site in the HIV genome; or A suitable amount of a composition comprising a vector encoding one or more of the isolated nucleic acid sequences; and a package insert, packaging material, sterile liquid, syringe and sterile container containing instructions for use A kit comprising one or more articles selected from the group consisting of:   24. The kit of claim 23, wherein the gRNA comprises a first gRNA complementary to a first target sequence in the HIV genome and a second gRNA complementary to a second target sequence in the HIV genome.   24. The kit according to claim 23, wherein the first target sequence is located in an LTR region in the HIV genome and the second target sequence is located in a structural gene and / or a nonstructural gene in the HIV genome. An expression vector for inactivating proviral DNA integrated into the genome of a host cell in which a retrovirus is latently infected,
At least one isolated nucleic acid sequence encoding a CRISPR-related endonuclease and at least one guide RNA (gRNA) complementary to a target sequence in proviral DNA is used for the host cell in which the retrovirus is latently infected. An expression vector which contains to inactivate proviral DNA integrated into the genome.   27. The at least gRNA comprises at least a first gRNA complementary to a target sequence in proviral DNA; and a second gRNA complementary to a second target sequence in the proviral DNA. Expression vector.   27. An expression vector according to claim 26, wherein the first target sequence is located in the LTR region of the HIV genome and the second target sequence is located in a structural gene and / or a non-structural gene of the HIV genome. . A polynucleotide for inactivating a retroviral genome integrated into the genome of a host cell to which the retrovirus is latently infected,
The retrovirus has at least one nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) related endonuclease and at least one guide RNA (gRNA) complementary to the target sequence in the integrated retroviral genome. A polynucleotide comprising for inactivating the retroviral genome integrated into the genome of a latently infected host cell. A composition for eliminating retroviruses in vitro or in vivo, comprising:
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) related endonuclease and an isolated nucleic acid sequence encoding at least one guide RNA (gRNA) complementary to a target sequence in the retroviral genome, A composition which is a human immunodeficiency virus (HIV).   The at least one gRNA comprises at least a first gRNA complementary to a target sequence in the HIV genome; and a second gRNA complementary to another target sequence in the HIV genome, whereby two gRNAs 32. The composition of claim 30, wherein intervening sequences between are removed.   32. The composition of claim 30, wherein the target nucleic acid sequence comprises one or more nucleic acid sequences in coding and non-coding nucleic acid sequences of the HIV genome.   The target sequence includes one or more nucleic acid sequences in the HIV genome, and the one or more nucleic acid sequences include a long terminal repeat (LTR) nucleic acid sequence, a nucleic acid sequence encoding a structural protein, a non-structure 35. The composition of claim 32, comprising a nucleic acid sequence encoding a protein, or a combination thereof.   A nucleic acid sequence wherein the sequence encoding a structural protein encodes Gag, Gag-Pol precursor, Pro (protease), reverse transcriptase (RT), integrase (In), Env or a combination thereof. 34. The composition of claim 33, comprising.   34. The composition of claim 33, wherein the sequence encoding a nonstructural protein comprises a nucleic acid sequence encoding a control protein, an accessory protein, or a combination thereof.   36. The composition of claim 35, wherein the control protein comprises Tat, Rev, or a combination thereof.   36. The composition of claim 35, wherein the accessory protein comprises Nef, Vpr, Vpu, Vif or combinations thereof.   32. The composition of claim 30, wherein the gRNA nucleic acid sequence comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NOs: 1-57.   40. The composition of claim 38, wherein the gRNA nucleic acid sequence comprises a nucleic acid sequence comprising SEQ ID NOs: 1-57.   Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) related endonucleases and isolated nucleic acid sequences encoding two guide RNAs (gRNAs), each complementary to different target sequences in the retroviral genome, in vitro or A composition for eliminating retroviruses in vivo.   At least one guide RNA (gRNA) comprising at least a first gRNA complementary to a target sequence in the HIV genome; and a second gRNA complementary to the target sequence in the HIV genome, whereby two 41. The composition of claim 40, wherein intervening sequences between gRNAs are removed.   41. The composition of claim 40, wherein the target nucleic acid sequence comprises one or more nucleic acid sequences in the coding and non-coding nucleic acid sequences in the HIV genome.   The target sequence includes one or more nucleic acid sequences in the HIV genome, and the one or more nucleic acid sequences include a long terminal repeat (LTR) nucleic acid sequence, a nucleic acid sequence encoding a structural protein, a non-structure 43. The composition of claim 42, comprising a nucleic acid sequence encoding a protein, or a combination thereof.   A nucleic acid sequence wherein the sequence encoding a structural protein encodes Gag, Gag-Pol precursor, Pro (protease), reverse transcriptase (RT), integrase (In), Env or a combination thereof. 44. The composition of claim 43, comprising.   44. The composition of claim 43, wherein the sequence encoding a nonstructural protein comprises a nucleic acid sequence encoding a regulatory protein, an accessory protein, or a combination thereof.   46. The composition of claim 45, wherein the control protein comprises Tat, Rev, or a combination thereof.   46. The composition of claim 45, wherein the accessory protein comprises Nef, Vpr, Vpu, Vif or combinations thereof.   41. The composition of claim 40, wherein the gRNA nucleic acid sequence comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NOs: 1-57.   49. The composition of claim 48, wherein the gRNA nucleic acid sequence comprises a nucleic acid sequence comprising SEQ ID NOs: 1-57.

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